Humanized monoclonal antibodies that specifically bind and/or neutralize Japanese encephalitis virus (JEV) and their use

ABSTRACT

Disclosed herein are isolated humanized monoclonal antibodies that specifically bind Japanese encephalitis virus (JEV) with a binding affinity of about 1.0 nM or less. Nucleic acids encoding these antibodies, expression vectors including these nucleic acid molecules, and isolated host cells that express the nucleic acid molecules are also disclosed. Methods of treating, preventing, and/or ameliorating JEV infection in a subject with JEV also are disclosed. Additionally, the antibodies can be used to detect JEV in a sample, and methods of diagnosing JEV infection, or confirming a diagnosis of JEV infection in a subject, are disclosed herein that utilize these antibodies.

CROSS REFERENCE TO RELATED REFERENCES

This is a continuation of U.S. patent application Ser. No. 12/937,227,filed Oct. 8, 2010, which is the U.S. national stage of PCT ApplicationNo. PCT/US2009/040227, filed Apr. 10, 2009, which was published inEnglish under PCT Article 21(2), which in turn claims the benefit ofU.S. Provisional Application No. 61/123,905, filed on Apr. 10, 2008. Theprior applications are incorporated herein by reference their entirety.

FIELD

This disclosure is related to humanized monoclonal antibodies,specifically to humanized monoclonal antibodies that specifically bindJapanese encephalitis virus (JEV), and their use.

BACKGROUND

Japanese encephalitis virus (JEV) is the prototype virus of the Japaneseencephalitis (JE) group belonging to the Flavivirus genus of theFlaviviridae family. Other members of the group include Kunjin virus,St. Louis encephalitis virus (SLEV) and West Nile encephalitis virus(WNV). JEV is widely distributed in South Asia, Southeast Asia, and theAsian Pacific Rim. In recent years, JE epidemics have spread topreviously unaffected areas, such as northern Australia (Hanna, et al.(1996) Med J Aust 165:256-60; Pyke, et al. (2001) Am J Trop Med Hyg65:747-53), Pakistan (Igarashi, et al. (1994) Microbiol Immunol38:827-30), India and Indonesia (Mackenzie, et al. (2004) Nat Med10:S98-109). The JE outbreak in India during July-November of 2005 wasthe longest and most severe in recent years, affecting in excess of5,000 persons and causing greater than 1,000 deaths (Parida, et al.(2006) Emerg Infect Dis 12:1427-30).

It is estimated that JEV causes 35,000-50,000 cases of encephalitis,including 10,000 deaths and as many neurologic sequelae each year (Tsai(2000) Vaccine 18 Suppl 2:1-25). Although only one JEV serotype is knownto exist, cross-neutralization experiments have demonstrated antigenicdifferences among JEV strains (Ali & Igarashi (1997) Microbiol Immunol41:241-52). Phylogenic studies have identified five JEV genotypes, fourof which are presently recognized (Chen, et al. (1992) Am J Trop Med Hyg47:61-9; Solomon, et al. (2003) J Virol 77:3091-8; Uchil &Satchidanandam (2001) Am J Trop Med Hyg 65:242-51). The widegeographical distribution and the existence of multiple strains, coupledwith the high rate of mortality and residual neurological complicationsin survivors, make JEV infection an important public health problem.

The JE-VAX® vaccine currently available in most countries is aninactivated whole virus vaccine prepared from virus grown in mouse brainand a three-dose regimen is required for young children (Monath (2002)Curr Top Microbiol Immunol 267:105-38). The requirements of multipledoses and the high vaccine manufacturing cost have prevented manycountries from adopting an effective JEV vaccination campaign. A liveattenuated vaccine, JEV strain SA14-14-2, has been developed in Chinaand was efficacious after one dose in a recent case-controlled study(Tandan, et al. (2007) Vaccine 25:5041-5). In addition, there is achimeric JEV vaccine constructed from the attenuated yellow fever 17Dstrain in a late experimental stage (Monath, et al. (2003) J Infect Dis188:1213-30). However, until a JEV vaccine becomes generally available,a need remains for short-term prevention and therapeutic intervention ofencephalitic JEV infections.

SUMMARY

Provided herein are isolated humanized monoclonal antibodies thatspecifically bind and/or neutralize Japanese encephalitis virus (JEV)envelope protein. The humanized monoclonal antibodies bind JEV with anaffinity constant (K_(d)) of about 1.0 nM or less. Also provided areisolated humanized monoclonal antibodies that specifically bind toLys₁₇₉ within a β-strand in domain I of the envelope protein, isolatedhumanized monoclonal antibodies that specifically bind Ile₁₂₆ within thesmall loop between d and e β-strands in domain II of the envelopeprotein, and isolated humanized monoclonal antibodies that specificallybind Gly₃₀₂ within amino acids 302-309 of domain III of the envelopeprotein. In some embodiments, the human monoclonal antibodies includeFab fragments that include chimpanzee FRs and CDRs. Further provided arecompositions including the JEV-specific antibodies, nucleic acidsencoding these antibodies, expression vectors including the nucleicacids, and isolated host cells that express the nucleic acids.

Also provided are pharmaceutical compositions that include the humanizedJEV-specific monoclonal antibodies.

The antibodies and compositions provided herein can be used for avariety of purposes, such as for treating or inhibiting the developmentof JEV infection in a subject. Thus, disclosed herein is a method oftreating a subject diagnosed with JEV infection or at risk fordeveloping JEV infection, the method including administering to thesubject a therapeutically effective amount of the humanized JEVantibody, thereby treating the subject.

The antibodies and compositions provided herein also can be used fordiagnosing or confirming the diagnosis of JEV infection in a subject.Thus, provided herein is a method of confirming the diagnosis of JEVinfection in a subject, the method including contacting a sample fromthe subject diagnosed with JEV infection with a human monoclonalantibody that specifically binds JEV, and detecting binding of theantibody to the sample. An increase in binding of the antibody to thesample relative to binding of the antibody to a control sample confirmsthe JEV infection diagnosis. In some embodiments, the method furtherincludes contacting a second antibody that specifically recognizes theJEV-specific antibody with the sample, and detecting binding of thesecond antibody.

Similarly, a method is provided herein for detecting JEV infection in asubject, including contacting a sample from the subject with a humanizedmonoclonal antibody described herein, and detecting binding of theantibody to the sample. An increase in binding of the antibody to thesample relative to a control sample detects JEV infection in thesubject. In some embodiments, the methods further include contacting asecond antibody that specifically recognizes the JEV-specific antibodywith the sample, and detecting binding of the second antibody.

The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying figures.

BIOLOGICAL DEPOSIT

Plasmids encoding the humanized antibodies A3, B2, and E3 were depositedin accordance with the Budapest Treaty with the American Type CultureCollection (ATCC) Patent Depository, 10801 University Blvd., Manassas,Va., 20110, on Apr. 7, 2008. The plasmid encoding the humanized antibodyA3 was deposited as ATCC No. PTA-9138. The plasmid encoding thehumanized antibody B2 was deposited as ATCC No. PTA-9139 and the plasmidencoding the humanized antibody E3 was deposited as ATCC No. PTA-9140.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. is a diagram showing the alignment of V_(H) and V_(L) sequencesof chimpanzee JEV-specific Fabs (SEQ ID NOS: 1-6). Fabs were groupedaccording to the panning strategy: Group 1 (panning with JEV SA14-14-2virions); Group 2 (panning with epitope masking); and Group 3 (panningwith a JEV E domain III-specific recombinant protein). Sequences of themost potently neutralizing Fab in each group are provided (V_(H) upperpanel and V_(L) lower panel). Framework regions (FR), complementaritydetermining regions (CDR), sequences identities (.) and deletions (-)are indicated.

FIG. 2. is a series of three graphs showing an analysis of Fab bindingto JEV SA14-14-2. Fabs A3 (FIG. 2A), B2 (FIG. 2B) and E3 (FIG. 2C) wereaffinity purified, biotinylated, and used for analysis of bindingactivity to JEV SA14-14-2 by competition ELISA in the presence ofcompeting, unlabeled Fabs. Fab 5H2, which did not react with JEV, wasused as a negative control. Fab 1A5 is a flavivirus cross-reactiveantibody that binds to determinants in the flavivirus E fusion loop. JEVFab clones were grouped according to the panning procedure: Group 1: A3;Group 2: B2, A8 and G1; and Group 3: E3 and B12.

FIG. 3. is a series of digital images of gels showing the identificationof E as the binding target of JEV MAbs. In FIG. 3A, purified MAbs wereincubated with nitrocellulose membranes blotted with JEV SA14-14-2strain (upper panel) or the recombinant JEV E protein (lower panel),separated by 4 to 12% SDS-PAGE under non-reducing conditions. HMAFagainst JEV was included as a positive control. In FIG. 3B,immunoprecipitation of JEV infected mosquito cell lysates (upper panel)or a recombinant JEV E protein (lower panel) was analyzed undernon-reducing conditions by Western blotting with MAb 6B4A-10 andanti-mouse IgG-HRP (upper panel) or with MAb anti-V5 epitope-HRP (lowerpanel). Molecular size markers are shown on the left.

FIG. 4. is a series of graphs showing the binding activities of JEVFabs. In FIG. 4A, binding in response to different concentrations ofpurified Fabs was analyzed by ELISA on JEV SA14-14-2 virions attached tothe solid phase with HMAF. The Fab concentration required to reach 50%saturation binding was calculated by nonlinear regression. FIG. 4B showsrepresentative sensograms of Fabs A3 (top panel) and B2 (bottom panel)analyzed by SPR with the recombinant JEV E protein, showing curvesfitted using a model for continuous ligand distributions, combined witha 2-compartment approximation of mass transport. Experimental data andfitted curves are shown.

FIG. 5. is a series of graphs showing neutralization of parental JEV andits variants using chimpanzee JEV Fabs. JEV neutralization-escapevariants v1, v2 and v3 were selected with the Fabs A3, B2, and E3,respectively. Neutralization titrations by PRNT against parental JEV andantigenic variants v1, v2 and v3 with Fabs A3 (FIG. 5A), B2 (FIG. 5B),and E3 (FIG. 5C) are shown. PRNT was performed using approximately 70FFU of each virus for incubation with serially diluted antibody at 37°C. for 1 hour. The reaction mixture was used to infect Vero Cells. Fociof infected cells were detected by immuno-staining.

FIG. 6. is an alignment and 3-D structure model showing the localizationof epitope determinants of JEV MAbs. FIG. 6A shows the alignment ofamino acid sequences among flavivirus E's. Sequences surrounding theamino acid substitutions found in JEV variants v1, v2 and v3 are shown.Clustal W v2 was used to obtain an optimal amino acid sequence alignmentfile. FIG. 6B shows the 3-D structure model of JEV SA14-14-2 E protein.The structure modeling was performed with the crystal coordinates of WNV(PDB code, 2169) and a Swiss Modeling Workstation. Molecular graphicsimages were produced using the UCSF Chimera program. Positions of126Ile, 136Lys, 179Lys, 219His and 302Gly as viewed from the top (upperpanel) and from the side (lower panel). E domain sequences are providedin grayscale: domain I includes 136Lys and 179Lys (dark font), domain IIincludes 126Ile and 219His (light font) and domain III includes 302Gly(dark font).

FIG. 7 is a series of digital images of gels showing the bindinganalysis of mutant E proteins containing a single substitution atposition 126, 136, 179, 219 or 302. FIG. 7A shows the binding ofhumanized MAbs A3, E3 (top panel), and the control mouse MAb anti-V5epitope (bottom panel) to various mutant proteins as analyzed by Westernblotting. The wild type (WT) and mutant E proteins reacted with theindicated antibody and developed with an HRP-conjugated secondaryantibody. FIG. 7B shows the binding of MAbs A3, B2 and E3 to WT andmutant E proteins (top panel) and the control mouse MAb 6B4A-10 (bottompanel) analyzed by immunoprecipitation in the absence of detergents. Theimmunoprecipitates were developed by Western blotting using MAb anti-V5epitope HRP-conjugate.

FIG. 8. is a series of graphs showing the protective activity ofhumanized JEV IgG1 antibodies (MAbs) using a mouse JEV challenge model.Inbred ddy mice (n=12) were injected i.p. with MAb A3 (FIG. 8A), MAb B2(FIG. 8B) or MAb E3 (FIG. 8C) at various doses indicated. Un-protectedcontrol mice were administrated PBS diluent. 24 hours later mice wereinfected i.c. with JEV strain.

SEQUENCE LISTING

The Sequence Listing is submitted as an ASCII text file(4239-80933-04_Sequence_Listing.txt, Jul. 12, 2013, 36.1 KB), which isincorporated by reference herein.

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. In the accompanying sequence listing:

SEQ ID NO: 1 is the amino acid sequence of the heavy chain of monoclonalantibody A3.

SEQ ID NO: 2 is the amino acid sequence of the heavy chain of monoclonalantibody B2.

SEQ ID NO: 3 is the amino acid sequence of the heavy chain of monoclonalantibody E3.

SEQ ID NO: 4 is the amino acid sequence of the light chain of monoclonalantibody A3.

SEQ ID NO: 5. is the amino acid sequence of the light chain ofmonoclonal antibody B2.

SEQ ID NO: 6 is the amino acid sequence of the light chain of monoclonalantibody E3.

SEQ ID NO: 7 is the nucleic acid sequence of a forward primer used toamplify the DNA encoding amino acids 131-692 of the PrM/N-terminal 80% Efusion protein from JEV cDNA.

SEQ ID NO: 8 is the nucleic acid sequence of a reverse primer used toamplify the DNA encoding amino acids 131-692 of the PrM/N-terminal 80% Efusion protein from JEV cDNA.

SEQ ID NO: 9 is the nucleic acid sequence encoding JEV ENV protein. SEQID NO: 10 is the amino acid sequence of the JEV ENV protein from JEVstrain SA14-14-2.

SEQ ID NO: 11 is the amino acid sequence of the Fc Region of human IgG.SEQ ID NOs: 12-30 are the amino acid sequences of a deletion at theN-terminal region of the C_(H)2 domain in the Fc region of human IgG.

SEQ ID NO: 31 is the full-length variable region amino acid sequence ofthe heavy chain of monoclonal antibody A3.

SEQ ID NO: 32 is the full-length variable region amino acid sequence ofthe heavy chain of monoclonal antibody B2.

SEQ ID NO: 33 is the full-length variable region amino acid sequence ofthe heavy chain of monoclonal antibody E3.

SEQ ID NO: 34 is the full-length variable region amino acid sequence ofthe light chain of monoclonal antibody A3.

SEQ ID NO: 35 is the full-length variable region amino acid sequence ofthe light chain of monoclonal antibody B2.

SEQ ID NO: 36 is the full-length variable region amino acid sequence ofthe light chain of monoclonal antibody E3.

DETAILED DESCRIPTION

I. Abbreviations

3SR: Self-sustained sequence replication

ADCC: Antibody-dependent cell-mediated cytotoxicity

AST: average survival time

BSA: bovine serum albumin

BRET: bioluminescence resonance energy transfer

C capsid

CDR: Complementarity determining region

DENY: dengue virus

DMEM: Dulbecco's Modified Essential Medium

DTE: dithioerythritol

E: envelope

ELISA: Enzyme-linked immunosorbent assay

EM: Effector molecule

FACS: Fluorescence-activated cell sorting

FBS: Fetal bovine serum

FFU: focus forming units

FITC: Fluorescein isothiocyanate

FRET: fluorescence resonance energy transfer

GFP: Green fluorescent protein

GPI: Glycosylphosphatidylinositol

HMAF: Hyperimmune mouse ascites fluid

HRP: Horseradish peroxidase

I.C.: Intracerebral

Ig: Immunoglobulin

I.P.: intraperitoneal

JEV: Japanese encephalitis virus

LCR: Ligase chain reaction

LDH: Lactate dehydrogenase

LGTV: Langat virus

mAb: Monoclonal antibody

MEM: Minimum Essential Medium

MOI: multiple of infection

MPBS: Milk/PBS

PAGE: Polyacrylamide gel electrophoresis

PBMC: Peripheral blood mononuclear cells

PBS: Phosphate-buffered saline

PBST: PBS-Tween 20

PCR: Polymerase chain reaction

prM/M: pre-membrane/membrane

PRNT: plaque reduction neutralization tests

PE: Pseudomonas exotoxin

PEG: Polyethylene glycol

RIA: Radioimmunoassay

S2: Schneider's Drosophila Line 2

SDS: Sodium dodecyl sulfate

SLEV: St. Louis encephalitis virus

SC: subcutaneous

SPR: surface plasmon resonance

TAS: transcription-based amplification

WNV: West Nile encephalitis virus

YFP: Yellow fluorescent protein

II. Abbreviations, Terms and Methods

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology canbe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of thedisclosure, the following explanations of specific terms are provided:

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects.

Antibody: A polypeptide ligand including at least a light chain or heavychain immunoglobulin variable region which specifically recognizes andbinds an epitope of an antigen, such as JEV envelope protein or afragment thereof. Antibodies are composed of a heavy and a light chain,each of which has a variable region, termed the variable heavy (V_(H))region and the variable light (V_(L)) region. Together, the V_(H) regionand the V_(L) region are responsible for binding the antigen recognizedby the antibody.

Antibodies include intact immunoglobulins and the variants and portionsof antibodies well known in the art, such as Fab fragments, Fab′fragments, F(ab)′₂ fragments, single chain Fv proteins (“scFv”), anddisulfide stabilized Fv proteins (“dsFv”). A scFv protein is a fusionprotein in which a light chain variable region of an immunoglobulin anda heavy chain variable region of an immunoglobulin are bound by alinker, while in dsFvs, the chains have been mutated to introduce adisulfide bond to stabilize the association of the chains. The term alsoincludes genetically engineered forms such as chimeric antibodies (forexample, humanized murine or humanized chimpanzee antibodies),heteroconjugate antibodies (such as, bispecific antibodies). See also,Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford,Ill.); Kuby, J., Immunology, 3^(rd) Ed., W. H. Freeman & Co., New York,1997.

Typically, a naturally occurring immunoglobulin has heavy (H) chains andlight (L) chains interconnected by disulfide bonds. There are two typesof light chain, lambda (λ) and kappa (k). There are five main heavychain classes (or isotypes) which determine the functional activity ofan antibody molecule: IgM, IgD, IgG, IgA and IgE.

Each heavy and light chain contains a constant region and a variableregion, (the regions are also known as “domains”). In combination, theheavy and the light chain variable regions specifically bind theantigen. Light and heavy chain variable regions contain a “framework”region interrupted by three hypervariable regions, also called“complementarity-determining regions” or “CDRs.” The extent of theframework region and CDRs has been defined (see, Kabat et al., Sequencesof Proteins of Immunological Interest, U.S. Department of Health andHuman Services, 1991, which is hereby incorporated by reference). TheKabat database is now maintained online. The sequences of the frameworkregions of different light or heavy chains are relatively conservedwithin a species, such as humans. The framework region of an antibody,that is the combined framework regions of the constituent light andheavy chains, serves to position and align the CDRs in three-dimensionalspace.

The CDRs are primarily responsible for binding to an epitope of anantigen. The CDRs of each chain are typically referred to as CDR1, CDR2,and CDR3, numbered sequentially starting from the N-terminus, and arealso typically identified by the chain in which the particular CDR islocated. Thus, a V_(H) CDR3 is located in the variable domain of theheavy chain of the antibody in which it is found, whereas a V_(L) CDR1is the CDR1 from the variable domain of the light chain of the antibodyin which it is found. An antibody that binds JEV will have a specificV_(H) region and the V_(L) region sequence, and thus specific CDRsequences. Antibodies with different specificities (i.e. differentcombining sites for different antigens) have different CDRs. Although itis the CDRs that vary from antibody to antibody, only a limited numberof amino acid positions within the CDRs are directly involved in antigenbinding. These positions within the CDRs are called specificitydetermining residues (SDRs).

References to “V_(H)” or “VH” refer to the variable region of animmunoglobulin heavy chain, including that of an Fv, scFv, dsFv or Fab.References to “V_(L)” or “VL” refer to the variable region of animmunoglobulin light chain, including that of an Fv, scFv, dsFv or Fab.

A “monoclonal antibody” is an antibody produced by a single clone ofB-lymphocytes or by a cell into which the light and heavy chain genes ofa single antibody have been transfected. Monoclonal antibodies areproduced by methods known to those of skill in the art, for instance bymaking hybrid antibody-forming cells from a fusion of myeloma cells withimmune spleen cells. Monoclonal antibodies include humanized monoclonalantibodies.

A “chimeric antibody” has framework residues from one species, such ashuman, and CDRs (which generally confer antigen binding) from anotherspecies, such as a chimpanzee antibody that specifically binds JEVand/or JEV envelope protein.

A “humanized” immunoglobulin is an immunoglobulin including a humanframework region and one or more CDRs (or SDRs) from a non-human (forexample a mouse, rat, chimpanzee or synthetic) immunoglobulin. Thenon-human immunoglobulin providing the CDRs is termed a “donor,” and thehuman immunoglobulin providing the framework is termed an “acceptor.” Inone embodiment, all the CDRs are from the donor immunoglobulin in ahumanized immunoglobulin. Constant regions need not be present, but ifthey are, they must be substantially identical to human immunoglobulinconstant regions, for instance, at least about 85-90%, such as about 95%or more identical. Hence, all parts of a humanized immunoglobulin,except possibly the CDRs, are substantially identical to correspondingparts of natural human immunoglobulin sequences.

A “humanized antibody” is an antibody comprising a humanized light chainand a humanized heavy chain immunoglobulin. A humanized antibody bindsto the same antigen as the donor antibody that provides the CDRs.“Humanized” forms of non-human (for instance, murine or chimpanzee)antibodies are chimeric antibodies that contain minimal sequence derivedfrom non-human immunoglobulin. For the most part, humanized antibodiesare human immunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate, such as chimpanzee, having thedesired specificity, affinity, and capacity. The acceptor framework of ahumanized immunoglobulin or antibody may have a limited number ofsubstitutions by amino acids taken from the donor framework. Humanizedor other monoclonal antibodies can have additional conservative aminoacid substitutions which have substantially no effect on antigen bindingor other immunoglobulin functions. In general, the humanized antibodywill include substantially all of at least one, and typically two,variable domains, in which all or substantially all of the hypervariableloops correspond to those of a non-human immunoglobulin and all orsubstantially all of the FR regions are those of a human immunoglobulinsequence. The humanized antibody optionally also will include at least aportion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin. In some embodiments, the C_(H)2 and/or C_(H)3domain is deleted or altered in the Fc region.

Humanized immunoglobulins can be constructed by means of geneticengineering (see for example, U.S. Pat. No. 5,585,089).

In one example, the framework and the CDRs are from the same originatinghuman heavy and/or light chain amino acid sequence. However, frameworksfrom one human antibody can be engineered to include CDRs from adifferent human antibody.

A “human” antibody (also called a “fully human” antibody) is an antibodythat includes human framework regions and all of the CDRs from a humanimmunoglobulin. In one example, the framework and the CDRs are from thesame originating human heavy and/or light chain amino acid sequence.However, frameworks from one human antibody can be engineered to includeCDRs from a different human antibody. All parts of a humanimmunoglobulin are substantially identical to corresponding parts ofnatural human immunoglobulin sequences.

Binding affinity: Affinity of an antibody for an antigen. In oneembodiment, affinity is calculated by a modification of the Scatchardmethod described by Frankel et al., Mol. Immunol., 16:101-106, 1979. Inanother embodiment, binding affinity is measured by an antigen/antibodydissociation rate. In another embodiment, a high binding affinity ismeasured by a competition radioimmunoassay. In another embodiment,binding affinity is measured by ELISA. In one embodiment, the antibodiesbind JEV with an affinity constant (K_(d)) of about 1.0 nM or less. Inseveral embodiments, the humanized monoclonal antibodies bind JEV with abinding affinity of about 0.95 nM or less, about 0.85 nM or less, about0.75 nM or less, about 0.65 nM or less, about 0.55 nM or less, about0.45 nM or less, about 0.35 nM or less, about 0.25 nM or less, or about0.15 nM or less. As used herein, a binding affinity of “about 1.0 nM”includes binding affinities of 0.9 to 1.1 nM. Similarly, a bindingaffinity of “about 0.5 nM” includes binding affinities of 0.4 to 0.6 nM.

Binding domain: The region of a polypeptide that binds to anothermolecule. In the case of an FcR, the binding domain can include aportion of a polypeptide chain thereof (for instance, the α chainthereof) that is responsible for binding an Fc region. One usefulbinding domain is the extracellular domain of an FcR α chain.

C_(H)2 domain: The “C_(H)2 domain” of a human IgG Fc region (alsoreferred to as “Cγ2” domain) usually extends from about amino acid 231to about amino acid 340. The C_(H)2 domain is unique in that it is notclosely paired with another domain. Rather, two N-linked branchedcarbohydrate chains are interposed between the two C_(H)2 domains of anintact native IgG molecule. It has been speculated that the carbohydratecan provide a substitute for the domain-domain pairing and helpstabilize the C_(H)2 domain.

C_(H)3 domain: The “C_(H)3 domain” includes the stretch of residuesC-terminal to a C_(H)2 domain in an Fc region (for instance, from aboutamino acid residue 341 to about amino acid residue 447 of an IgG). See,for instance, SEQ ID NO: 11.

Chimeric antibody: An antibody that includes sequences derived from twodifferent antibodies, which typically are of different species. Chimericantibodies can include human and murine antibody domains, generallyhuman constant regions and murine variable regions, murine CDRs and/ormurine SDRs. In other examples, chimeric antibodies include human andchimpanzee antibody domains, generally human constant regions andchimpanzee variable regions, chimpanzee CDRs and/or chimpanzee SDRs.

Conservative variants: “Conservative” amino acid substitutions are thosesubstitutions that do not substantially affect or decrease the affinityof an antibody to JEV. For example, a human antibody that specificallybinds JEV can include at most about 1, at most about 2, at most about 5,and most about 10, or at most about 15 conservative substitutions andspecifically bind the original JEV polypeptide. The term conservativevariation also includes the use of a substituted amino acid in place ofan unsubstituted parent amino acid, provided that antibody specificallybinds JEV. Non-conservative substitutions are those that reduce anactivity or binding to JEV or JEV envelope protein.

Conservative amino acid substitution tables providing functionallysimilar amino acids are well known to one of ordinary skill in the art.The following six groups are examples of amino acids that are consideredto be conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Complementarity determining region (CDR): Amino acid sequences whichtogether define the binding affinity and specificity of the natural Fvregion of a native Ig binding site. The light and heavy chains of an Igeach have three CDRs, designated L-CDR1, L-CDR2, L-CDR3 and H-CDR1,H-CDR2, H-CDR3, respectively.

Contacting: Placement in direct physical association; includes both insolid and liquid form.

Degenerate variant: A polynucleotide encoding a JEV polypeptide or anantibody that binds JEV that includes a sequence that is degenerate as aresult of the genetic code. There are 20 natural amino acids, most ofwhich are specified by more than one codon. Therefore, all degeneratenucleotide sequences are included as long as the amino acid sequence ofthe JEV polypeptide or antibody that binds JEV encoded by the nucleotidesequence is unchanged.

Epitope: An antigenic determinant. These are particular chemical groupsor peptide sequences on a molecule that are antigenic, for instance,that elicit a specific immune response. An antibody specifically binds aparticular antigenic epitope on a polypeptide, such as JEV envelopeprotein.

Envelope protein: A membrane that coats the capsid layer of a virus. Acapsid is the protein shell of a virus. It includes of severaloligomeric subunits made of protein. The capsid encloses the geneticmaterial of the virus.

Capsids are broadly classified according to their structure. Themajority of viruses have capsids with either helical or icosahedralstructure. Some viruses, such as bacteriophages, have developed morecomplicated structures. The icosahedral shape, which has 20 equilateraltriangular faces, approximates a sphere, while the helical shape iscylindrical. The capsid faces may consist of one or more proteins. Forexample, the foot-and-mouth disease virus capsid has faces consisting ofthree proteins named VP1-3.

Some viruses are enveloped, meaning that the capsid is coated with alipid membrane known as the viral envelope. The envelope is acquired bythe capsid from an intracellular membrane in the virus' host; examplesof such intracellular membranes include the inner nuclear membrane, thegolgi membrane, and the cell's outer membrane.

Expressed: Translation of a nucleic acid into a protein. Proteins can beexpressed and remain intracellular, become a component of the cellsurface membrane, or be secreted into the extracellular matrix ormedium.

Fc region: The term “Fc region” includes a C-terminal region of animmunoglobulin heavy chain. The “Fc region” can be a native sequence Fcregion or a variant Fc region. Although the boundaries of the Fc regionof an immunoglobulin heavy chain might vary, the human IgG heavy chainFc region is usually defined to stretch from an amino acid residue atposition Cys226, or from Pro230, to the carboxyl-terminus thereof. TheFc region of an immunoglobulin generally includes two constant domains,C_(H)2 and C_(H)3.

A “functional Fc region” possesses an “effector function” of a nativesequence Fc region. Exemplary “effector functions” include C1q binding;complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity; phagocytosis; downregulation of cell surface receptors (for instance, B cell receptor),etc. Such effector functions generally require the Fc region to becombined with a binding domain (for instance, an antibody variabledomain) and can be assessed using various assays as herein disclosed,for example.

A “native sequence Fc region” includes an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature. Nativesequence human Fc regions include a native sequence human IgG1 Fcregion; native sequence human IgG2 Fc region; native sequence human IgG3Fc region; and native sequence human IgG4 Fc region as well as naturallyoccurring variants thereof. In one specific, non-limiting example, ahuman Fc region amino acid sequence is

(SEQ ID NO: 11) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVDHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A “variant Fc region” includes an amino acid sequence that differs fromthat of a native sequence Fc region by virtue of at least one “aminoacid modification” as herein defined. Generally, the variant Fc regionhas at least one amino acid modification compared to a native sequenceFc region or to the Fc region of a parent polypeptide, for instance,from about one to about ten amino acid modifications in a nativesequence Fc region or in the Fc region of the parent polypeptide.Embodiments disclosed herein include variant Fc regions that can havethe following degrees of amino acid sequence homology or identity to theFc region of a parent polypeptide, for example: 35%, 36%, 37%, 38%, 39%,40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99%. Candidate variant Fc regions having greater thanor equal to 35% homology or identity can be identified by methods knownin the art and can be subsequently examined using functional assays, forexample, the assays described herein and those known in the art. Thevariant Fc regions described herein, in some examples, possess at leastabout 80% homology with a native sequence Fc region and/or with an Fcregion of a parent polypeptide, and most preferably at least about 90%homology therewith, more preferably at least about 95% homologytherewith.

The term “Fc region-containing polypeptide” refers to a polypeptide,such as an antibody, that includes an Fc region.

The terms “Fc receptor” or “FcR” are used to describe a receptor thatbinds to the Fc region of an antibody. In one example, an FcR is anative sequence human FcR. In other examples, an FcR is one that bindsan IgG antibody (a gamma receptor) and includes receptors of the FcγRI,FcγRII, and FcγRIII subclasses, including allelic variants andalternatively spliced forms of these receptors. FcγRII receptors includeFcγRIIA (an “activating receptor”) and FcγRIIB (an “inhibitingreceptor”), which have similar amino acid sequences that differprimarily in the cytoplasmic domains thereof. Activating receptorFcγRIIA contains an immunoreceptor tyrosine-based activation motif inits cytoplasmic domain. Inhibiting receptor FcγRIIB contains animmunoreceptor tyrosine-based inhibition motif in its cytoplasmicdomain. Other FcRs, including those to be identified in the future, areencompassed by the term “FcR” as used herein. The term also includes theneonatal receptor, FcRn, which is responsible for the transfer ofmaternal IgGs to the fetus. The term includes allotypes, such asFcγRIIIA allotypes: FcγRIIIA-Phe₁₅₈, FcγRIIIA-Val₁₅₈, FcγRIIA-R₁₃₁and/or FcγRIIA-H₁₃₁.

“Human effector cells” are leukocytes that express one or more FcRs andperform effector functions.

A polypeptide variant with “altered” FcR binding affinity is one thathas diminished FcR binding activity compared to a parent polypeptide orto a polypeptide including a native sequence Fc region. The polypeptidevariant that “displays decreased binding” to an FcR binds at least oneFcR with worse affinity than a parent polypeptide. The decrease inbinding compared to a parent polypeptide can be about 40% or moredecrease in binding, for instance, down to a variant that possesseslittle or no appreciable binding to the FcR. Such variants that displaydecreased binding to an FcR can possess little or no appreciable bindingto an FcR, for instance, 0-20% binding to the FcR compared to a nativesequence IgG Fc region.

Framework region: Amino acid sequences interposed between CDRs.Framework regions include variable light and variable heavy frameworkregions. The framework regions serve to hold the CDRs in an appropriateorientation for antigen binding.

Hinge region: A region stretching from Glu216 to Pro230 of human IgG1.Hinge regions of other IgG isotypes can be aligned with the IgG1sequence by placing the first and last cysteine residues forminginter-heavy chain S—S bonds in the same positions. These regions arewell known in the art.

Host cells: Cells in which a vector can be propagated and its DNAexpressed. The cell can be prokaryotic or eukaryotic. The term alsoincludes any progeny of the subject host cell. It is understood that allprogeny may not be identical to the parental cell since there can bemutations that occur during replication. However, such progeny areincluded when the term “host cell” is used.

Hypervariable region: The amino acid residues of an antibody that areresponsible for antigen-binding. The hypervariable region includes aminoacid residues from a “complementarity determining region” or “CDR” (forinstance, residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the lightchain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in theheavy chain variable domain and/or those residues from a “hypervariableloop” (for instance, residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) inthe light chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101(H3) in the heavy chain variable domain. “Framework” or “FR” residuesare those variable domain residues other than the hypervariable regionresidues as herein defined.

Immune response: A response of a cell of the immune system, such as a Bcell, T cell, or monocyte, to a stimulus. In one embodiment, theresponse is specific for a particular antigen (an “antigen-specificresponse”). In one embodiment, an immune response is a T cell response,such as a CD4+ response or a CD8+ response.

In another embodiment, the response is a B cell response, and results inthe production of specific antibodies.

Immunogenic peptide: A peptide such as an envelope protein whichincludes an allele-specific motif or other sequence, such as anN-terminal repeat, such that the peptide will bind an MHC molecule andinduce a cytotoxic T lymphocyte (“CTL”) response, or a B cell response(for example, antibody production) against the antigen from which theimmunogenic peptide is derived.

In one embodiment, immunogenic peptides are identified using sequencemotifs or other methods, such as neural net or polynomialdeterminations, known in the art. Typically, algorithms are used todetermine the “binding threshold” of peptides to select those withscores that give them a high probability of binding at a certainaffinity and will be immunogenic. The algorithms are based either on theeffects on MHC binding of a particular amino acid at a particularposition, the effects on antibody binding of a particular amino acid ata particular position, or the effects on binding of a particularsubstitution in a motif-containing peptide. Within the context of animmunogenic peptide, a “conserved residue” is one which appears in asignificantly higher frequency than would be expected by randomdistribution at a particular position in a peptide. In one embodiment, aconserved residue is one where the MHC structure can provide a contactpoint with the immunogenic peptide. In one specific non-limitingexample, an immunogenic polypeptide includes a region of JEV envelopeprotein, or a fragment thereof.

Immunogenic composition: A composition including a JEV polypeptide, suchas an envelope protein that induces a measurable CTL response againstcells expressing JEV polypeptide, or induces a measurable B cellresponse (such as production of antibodies) against a JEV polypeptide.It further refers to isolated nucleic acids encoding a JEV polypeptidethat can be used to express the JEV polypeptide (and thus be used toelicit an immune response against this polypeptide). For in vitro use,an immunogenic composition can consist of the isolated protein orpeptide epitope. For in vivo use, the immunogenic composition willtypically include the protein or immunogenic peptide in pharmaceuticallyacceptable carriers, and/or other agents. Any particular peptide, suchas a JEV polypeptide, or nucleic acid encoding the polypeptide, can bereadily tested for its ability to induce a CTL or B cell response byart-recognized assays. Immunogenic compositions can include adjuvants,which are well known to one of skill in the art.

Immunologically reactive conditions: Includes reference to conditionswhich allow an antibody raised against a particular epitope to bind tothat epitope to a detectably greater degree than, and/or to thesubstantial exclusion of, binding to substantially all other epitopes.Immunologically reactive conditions are dependent upon the format of theantibody binding reaction and typically are those utilized inimmunoassay protocols or those conditions encountered in vivo. SeeHarlow & Lane, supra, for a description of immunoassay formats andconditions. The immunologically reactive conditions employed in themethods are “physiological conditions” which include reference toconditions (such as temperature, osmolarity, and pH) that are typicalinside a living mammal or a mammalian cell. While it is recognized thatsome organs are subject to extreme conditions, the intra-organismal andintracellular environment normally lies around pH 7 (for instance, frompH 6.0 to pH 8.0, more typically pH 6.5 to 7.5), contains water as thepredominant solvent, and exists at a temperature above 0° C. and below50° C. Osmolarity is within the range that is supportive of cellviability and proliferation.

Isolated: An “isolated” biological component, such as a nucleic acid,protein (including antibodies) or organelle, has been substantiallyseparated or purified away from other biological components in theenvironment (such as a cell) in which the component naturally occurs,for instance, other chromosomal and extra-chromosomal DNA and RNA,proteins and organelles. Nucleic acids and proteins that have been“isolated” include nucleic acids and proteins purified by standardpurification methods. The term also embraces nucleic acids and proteinsprepared by recombinant expression in a host cell as well as chemicallysynthesized nucleic acids.

Japanese encephalitis virus (JEV): The virus that causes themosquito-borne disease Japanese encephalitis. JEV is a virus from thefamily Flaviviridae. Domestic pigs and wild birds are reservoirs of thevirus; transmission to humans may cause severe symptoms. One of the mostimportant vectors of this disease is the mosquito Culextritaeniorhynchus. This disease is most prevalent in Southeast Asia andthe Far East.

JEV is an enveloped virus of the genus flavivirus; it is closely relatedto the West Nile virus and St. Louis encephalitis virus. Positive sensesingle stranded RNA genome is packaged in the capsid, formed by thecapsid protein. The outer envelope is formed by envelope (E) protein andis the protective antigen. The genome also encodes several nonstructuralproteins (NS1, NS2a, NS2 b, NS3, N4a, NS4b, NS5). NS1 is produced assecretory form also. NS3 is a putative helicase, and NS5 is the viralpolymerase. It has been noted that the Japanese encephalitis virus (JEV)infects the lumen of the endoplasmic reticulum and rapidly accumulatessubstantial amounts of viral proteins for the JEV.

Label: A detectable compound or composition that is conjugated directlyor indirectly to another molecule, such as an antibody or a protein, tofacilitate detection of that molecule. Specific, non-limiting examplesof labels include fluorescent tags, enzymatic linkages, and radioactiveisotopes. In one example, a “labeled antibody” refers to incorporationof another molecule in the antibody. For example, the label is adetectable marker, such as the incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotinyl moieties that can bedetected by marked avidin (for example, streptavidin containing afluorescent marker or enzymatic activity that can be detected by opticalor colorimetric methods). Various methods of labeling polypeptides andglycoproteins are known in the art and can be used. Examples of labelsfor polypeptides include, but are not limited to, the following:radioisotopes or radionucleotides (such as ³⁵S or ¹³¹I) fluorescentlabels (such as fluorescein isothiocyanate (FITC), rhodamine, lanthanidephosphors), enzymatic labels (such as horseradish peroxidase,beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescentmarkers, biotinyl groups, predetermined polypeptide epitopes recognizedby a secondary reporter (such as a leucine zipper pair sequences,binding sites for secondary antibodies, metal binding domains, epitopetags), or magnetic agents, such as gadolinium chelates. In someembodiments, labels are attached by spacer arms of various lengths toreduce potential steric hindrance.

Linker: In some cases, a linker is a peptide within an antibody bindingfragment (such as an Fv fragment) which serves to indirectly bond thevariable heavy chain to the variable light chain. “Linker” can alsorefer to a peptide serving to link a targeting moiety, such as anantibody, to an effector molecule, such as a cytotoxin or a detectablelabel.

The terms “conjugating,” “joining,” “bonding” or “linking” refer tomaking two polypeptides into one contiguous polypeptide molecule, or tocovalently attaching a radionuclide or other molecule to a polypeptide,such as an scFv. In the specific context, the terms include reference tojoining a ligand, such as an antibody moiety, to an effector molecule,such as a label. The linkage can be either by chemical or recombinantmeans. “Chemical means” refers to a reaction between the antibody moietyand the effector molecule such that there is a covalent bond formedbetween the two molecules to form one molecule.

Mammal: This term includes both human and non-human mammals. Similarly,the term “subject” includes both human and veterinary subjects.

Major histocompatibility complex (MHC): Generic designation meant toencompass the histocompatibility antigen systems described in differentspecies, including the human leukocyte antigens (“HLA”). The term“motif” refers to the pattern of residues in a peptide of definedlength, usually about 8 to about 11 amino acids, which is recognized bya particular MHC allele. The peptide motifs are typically different foreach MHC allele and differ in the pattern of the highly conservedresidues and negative binding residues.

Nucleic acid: A polymer composed of nucleotide units (ribonucleotides,deoxyribonucleotides, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof) linked viaphosphodiester bonds, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof. Thus, the termincludes nucleotide polymers in which the nucleotides and the linkagesbetween them include non-naturally occurring synthetic analogs, such as,for example and without limitation, phosphorothioates, phosphoramidates,methyl phosphonates, chiral-methyl phosphonates, 2-O-methylribonucleotides, peptide-nucleic acids (PNAs), and the like. Suchpolynucleotides can be synthesized, for example, using an automated DNAsynthesizer. The term “oligonucleotide” typically refers to shortpolynucleotides, generally no greater than about 50 nucleotides. It willbe understood that when a nucleotide sequence is represented by a DNAsequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e.,A, U, G, C) in which “U” replaces “T.”

Conventional notation is used herein to describe nucleotide sequences:the left-hand end of a single-stranded nucleotide sequence is the5′-end; the left-hand direction of a double-stranded nucleotide sequenceis referred to as the 5′-direction. The direction of 5′ to 3′ additionof nucleotides to nascent RNA transcripts is referred to as thetranscription direction. The DNA strand having the same sequence as anmRNA is referred to as the “coding strand;” sequences on the DNA strandhaving the same sequence as an mRNA transcribed from that DNA and whichare located 5′ to the 5′-end of the RNA transcript are referred to as“upstream sequences;” sequences on the DNA strand having the samesequence as the RNA and which are 3′ to the 3′ end of the coding RNAtranscript are referred to as “downstream sequences.”

“cDNA” refers to a DNA that is complementary or identical to an mRNA, ineither single stranded or double stranded form.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(for instance, rRNA, tRNA and mRNA) or a defined sequence of amino acidsand the biological properties resulting therefrom. Thus, a gene encodesa protein if transcription and translation of mRNA produced by that geneproduces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and non-codingstrand, used as the template for transcription, of a gene or cDNA can bereferred to as encoding the protein or other product of that gene orcDNA. Unless otherwise specified, a “nucleotide sequence encoding anamino acid sequence” includes all nucleotide sequences that aredegenerate versions of each other and that encode the same amino acidsequence. Nucleotide sequences that encode proteins and RNA can includeintrons.

“Recombinant nucleic acid” refers to a nucleic acid having nucleotidesequences that are not naturally joined together. This includes nucleicacid vectors including an amplified or assembled nucleic acid which canbe used to transform a suitable host cell. A host cell that includes therecombinant nucleic acid is referred to as a “recombinant host cell.”The gene is then expressed in the recombinant host cell to produce, suchas a “recombinant polypeptide.” A recombinant nucleic acid can serve anon-coding function (such as a promoter, origin of replication,ribosome-binding site, etc.) as well.

A first sequence is an “antisense” with respect to a second sequence ifa polynucleotide whose sequence is the first sequence specificallyhybridizes with a polynucleotide whose sequence is the second sequence.

Terms used to describe sequence relationships between two or morenucleotide sequences or amino acid sequences include “referencesequence,” “selected from,” “comparison window,” “identical,”“percentage of sequence identity,” “substantially identical,”“complementary,” and “substantially complementary.”

For sequence comparison of nucleic acid sequences, typically onesequence acts as a reference sequence, to which test sequences arecompared. When using a sequence comparison algorithm, test and referencesequences are entered into a computer, subsequence coordinates aredesignated, if necessary, and sequence algorithm program parameters aredesignated. Default program parameters are used. Methods of alignment ofsequences for comparison are well known in the art. Various programs andalignment algorithms are described in: Smith and Waterman, Adv. Appl.Math. 2:482, 1981; Needleman and Wunsch, J. Mol. Biol. 48:443, 1970;Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988; Higginsand Sharp, Gene 73:237, 1988; Higgins and Sharp, CABIOS 5:151, 1989; andCorpet et al., Nucleic Acids Research 16:10881, 1988.

One example of a useful algorithm is PILEUP. PILEUP uses asimplification of the progressive alignment method of Feng & Doolittle,J. Mol. Evol. 35:351-360, 1987. The method used is similar to the methoddescribed by Higgins & Sharp, CABIOS 5:151-153, 1989. Using PILEUP, areference sequence is compared to other test sequences to determine thepercent sequence identity relationship using the following parameters:default gap weight (3.00), default gap length weight (0.10), andweighted end gaps. PILEUP can be obtained from the GCG sequence analysissoftware package, such as version 7.0 (Devereaux et al., Nuc. Acids Res.12:387-395, 1984.

Oligonucleotide: A linear polynucleotide sequence of up to about 100nucleotide bases in length.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter, such as the CMV promoter, isoperably linked to a coding sequence if the promoter affects thetranscription or expression of the coding sequence. Generally, operablylinked DNA sequences are contiguous and, where necessary to join twoprotein-coding regions, in the same reading frame.

ORF (open reading frame): A series of nucleotide triplets (codons)coding for amino acids without any termination codons. These sequencesare usually translatable into a peptide.

Passive immunization: A type of immunization in which pre-made elementsof the immune system (for instance, antibodies) are transferred to asubject. Passive immunization occurs naturally during the transfer ofantibodies from mother to fetus during pregnancy. Artificial passiveimmunization is normally administered parenterally, and is particularlyeffective as both a therapeutic and a preventative strategy during anoutbreak of a particular disease or as an emergency treatment to toxins(for example, for tetanus). Passive immunization also is used when thereis a high risk of infection and insufficient time for the body todevelop its own immune response, or to reduce the symptoms of ongoing orimmunosuppressive diseases.

Artificially acquired passive immunity is a short-term immunizationachieved by the transfer of antibodies, which can be administered inseveral forms; as human or animal blood plasma or serum, as pooled humanimmunoglobulin for intravenous (IVIG) or intramuscular (IG) use, ashigh-titer human IVIG or IG from immunized or from donors recoveringfrom the disease, and as monoclonal antibodies (MAb). Passive transferis used prophylactically in the case of immunodeficiency diseases, suchas hypogammaglobulinemia. It is also used in the treatment of severaltypes of acute infection, and to treat poisoning. Immunity derived frompassive immunization lasts for only a short period of time, and there isalso a potential risk for hypersensitivity reactions, and serumsickness, especially from gamma globulin of non-human origin. Passiveimmunity provides immediate protection, but the body does not developmemory, therefore the patient is at risk of being infected by the samepathogen later. Methods of passive immunization are discussed in greaterdetail below in the Detailed Description.

Pharmaceutical agent: A chemical compound or composition capable ofinducing a desired therapeutic or prophylactic effect when properlyadministered to a subject or a cell.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers of use are conventional. Remington's Pharmaceutical Sciences,by E. W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition, 1975,describes compositions and formulations suitable for pharmaceuticaldelivery of the fusion proteins herein disclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually include injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (such as powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

Polynucleotide: The term polynucleotide or nucleic acid sequence refersto a polymeric form of nucleotide at least 10 bases in length. Arecombinant polynucleotide includes a polynucleotide that is notimmediately contiguous with both of the coding sequences with which itis immediately contiguous (one on the 5′ end and one on the 3′ end) inthe naturally occurring genome of the organism from which it is derived.The term therefore includes, for example, a recombinant DNA which isincorporated into a vector; into an autonomously replicating plasmid orvirus; or into the genomic DNA of a prokaryote or eukaryote, or whichexists as a separate molecule (such as a cDNA) independent of othersequences. The nucleotides can be ribonucleotides, deoxyribonucleotides,or modified forms of either nucleotide. The term includes single- anddouble-stranded forms of DNA.

Polypeptide: Any chain of amino acids, regardless of length orpost-translational modification (such as glycosylation orphosphorylation). In one embodiment, the polypeptide is JEV envelopepolypeptide. A “residue” refers to an amino acid or amino acid mimeticincorporated in a polypeptide by an amide bond or amide bond mimetic. Apolypeptide has an amino terminal (N-terminal) end and a carboxyterminal (C-terminal) end.

Preventing, treating or ameliorating a disease: “Preventing” a diseaserefers to inhibiting the full development of a disease. “Treating”refers to a therapeutic intervention that ameliorates a sign or symptomof a disease or pathological condition after it has begun to develop.“Ameliorating” refers to the reduction in the number or severity ofsigns or symptoms of a disease, such as JEV infection.

Promoter: A promoter is an array of nucleic acid control sequences thatdirects transcription of a nucleic acid. A promoter includes necessarynucleic acid sequences near the start site of transcription, forexample, in the case of a polymerase II type promoter, a TATA element. Apromoter also optionally includes distal enhancer or repressor elementswhich can be located as much as several thousand base pairs from thestart site of transcription. Both constitutive and inducible promotersare included (see for example, Bitter et al., Methods in Enzymology153:516-544, 1987).

Specific, non-limiting examples of promoters include promoters derivedfrom the genome of mammalian cells (such as the metallothioneinpromoter) or from mammalian viruses (such as the retrovirus longterminal repeat; the adenovirus late promoter; the vaccinia virus 7.5Kpromoter) can be used. Promoters produced by recombinant DNA orsynthetic techniques can also be used. A polynucleotide can be insertedinto an expression vector that contains a promoter sequence whichfacilitates the efficient transcription of the inserted genetic sequenceof the host. The expression vector typically contains an origin ofreplication, a promoter, as well as specific nucleic acid sequences thatallow phenotypic selection of the transformed cells.

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified peptidepreparation is one in which the peptide or protein is more enriched thanthe peptide or protein is in its natural environment within a cell. Inone embodiment, a preparation is purified such that the protein orpeptide represents at least 50% of the total peptide or protein contentof the preparation.

The JEV polypeptides disclosed herein, or antibodies that specificallybind JEV or a JEV polypeptide such as a envelope polypeptide, can bepurified by any of the means known in the art. See for example Guide toProtein Purification, ed. Deutscher, Meth. Enzymol. 185, Academic Press,San Diego, 1990; and Scopes, Protein Purification: Principles andPractice, Springer Verlag, New York, 1982. Substantial purificationdenotes purification from other proteins or cellular components. Asubstantially purified protein is at least 60%, 70%, 80%, 90%, 95% or98% pure. Thus, in one specific, non-limiting example, a substantiallypurified protein is 90% free of other proteins or cellular components.

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination is often accomplished by chemical synthesisor, more commonly, by the artificial manipulation of isolated segmentsof nucleic acids, for example, by genetic engineering techniques.

Sequence identity: The similarity between amino acid sequences isexpressed in terms of the similarity between the sequences, otherwisereferred to as sequence identity. Sequence identity is frequentlymeasured in terms of percentage identity (or similarity or homology);the higher the percentage, the more similar the two sequences are.Homologs or variants of a polypeptide will possess a relatively highdegree of sequence identity when aligned using standard methods.

Methods of alignment of sequences for comparison are well known in theart. Optimal alignment of sequences for comparison can be conducted, forexample, by the local homology algorithm of Smith & Waterman, Adv. Appl.Math. 2:482, 1981, by the homology alignment algorithm of Needleman &Wunsch, J. Mol. Biol. 48:443, 1970, by the search for similarity methodof Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444, 1988, bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by manual alignment andvisual inspection (see for example, Current Protocols in MolecularBiology (Ausubel et al., eds 1995 supplement).

Another example of algorithms that are suitable for determining percentsequence identity and sequence similarity are the National Center forBiotechnology Information (NCBI) Basic Local Alignment Search Tool(BLAST) and the BLAST 2.0 algorithm, which are described in Altschul etal., J. Mol. Biol. 215:403-410, 1990 and Altschul et al., Nucleic AcidsRes. 25:3389-3402, 1997. The BLAST tool is available from severalsources, including the National Center for Biotechnology Information(NCBI, Bethesda, Md.) and on the internet(http://www.ncbi.nlm.nih.gov/), for use in connection with the sequenceanalysis programs such as BLASTP, BLASTN, BLASTX, TBLASTN and TBLASTX.For example, the BLASTN program (for nucleotide sequences) uses asdefaults a word length (W) of 11, alignments (B) of 50, expectation (E)of 10, M=5, N=−4, and a comparison of both strands. In another example,the BLASTP program (for amino acid sequences) uses as defaults a wordlength (W) of 3, and expectation (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915,1989). A description of how to determine sequence identity using thisprogram is available on the NCBI website on the internet.

Homologs and variants of a V_(L) or a V_(H) of an antibody thatspecifically binds JEV or a JEV envelope polypeptide are typicallycharacterized by possession of at least about 75%, for example at leastabout 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity counted overthe full length alignment with the amino acid sequence of the antibodyusing the NCBI Blast 2.0, gapped blastp set to default parameters. Forcomparisons of amino acid sequences of greater than about 30 aminoacids, the Blast 2 sequences function is employed using the defaultBLOSUM62 matrix set to default parameters, (gap existence cost of 11,and a per residue gap cost of 1). When aligning short peptides (fewerthan around 30 amino acids), the alignment should be performed using theBlast 2 sequences function, employing the PAM30 matrix set to defaultparameters (open gap 9, extension gap 1 penalties). Proteins with evengreater similarity to the reference sequences will show increasingpercentage identities when assessed by this method, such as at least80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least99% sequence identity. When less than the entire sequence is beingcompared for sequence identity, homologs and variants will typicallypossess at least 80% sequence identity over short windows of 10-20 aminoacids, and can possess sequence identities of at least 85% or at least90% or 95% depending on their similarity to the reference sequence.Methods for determining sequence identity over such short windows areavailable at the NCBI website on the internet. One of skill in the artwill appreciate that these sequence identity ranges are provided forguidance only; it is entirely possible that strongly significanthomologs could be obtained that fall outside of the ranges provided.

Specific binding agent: An agent that binds substantially only to adefined target. Thus, a JEV specific binding agent is an agent thatbinds substantially to a JEV polypeptide. In one embodiment, thespecific binding agent is a humanized monoclonal antibody thatspecifically binds the JEV polypeptide.

The term “specifically binds” refers, with respect to an antigen such asJEV envelope protein, to the preferential association of an antibody orother ligand, in whole or part, with a cell or tissue bearing thatantigen and not to cells or tissues lacking that antigen. It isrecognized that a certain degree of non-specific interaction can occurbetween a molecule and a non-target cell or tissue. Nevertheless,specific binding can be distinguished as mediated through specificrecognition of the antigen. Although selectively reactive antibodiesbind antigen, they can do so with low affinity. On the other hand,specific binding results in a much stronger association between theantibody (or other ligand) and cells bearing the antigen than betweenthe bound antibody (or other ligand) and cells lacking the antigen.Specific binding typically results in greater than 2-fold, such asgreater than 5-fold, greater than 10-fold, or greater than 100-foldincrease in amount of bound antibody or other ligand (per unit time) toa cell or tissue bearing the JEV polypeptide as compared to a cell ortissue lacking the polypeptide. Specific binding to a protein under suchconditions requires an antibody that is selected for its specificity fora particular protein. A variety of immunoassay formats are appropriatefor selecting antibodies or other ligands specifically immunoreactivewith a particular protein. For example, solid-phase ELISA immunoassaysare routinely used to select monoclonal antibodies specificallyimmunoreactive with a protein. See Harlow & Lane, Antibodies, ALaboratory Manual, Cold Spring Harbor Publications, New York (1988), fora description of immunoassay formats and conditions that can be used todetermine specific immunoreactivity.

Subject: Living multi-cellular vertebrate organisms, a category thatincludes both human and veterinary subjects, including human andnon-human mammals.

Therapeutically effective amount: A quantity of a specific substancesufficient to achieve a desired effect in a subject being treated. Forinstance, this can be the amount necessary to inhibit or suppress thedevelopment of an infection, or to substantially reduce the symptoms ofan infection, for instance a JEV infection. In one embodiment, atherapeutically effective amount is the amount necessary to eliminate aJEV infection. When administered to a subject, a dosage will generallybe used that will achieve target tissue concentrations that has beenshown to achieve a desired in vitro effect.

Transduced: A transduced cell is a cell into which has been introduced anucleic acid molecule by molecular biology techniques. As used herein,the term transduction encompasses all techniques by which a nucleic acidmolecule might be introduced into such a cell, including transfectionwith viral vectors, transformation with plasmid vectors, andintroduction of naked DNA by electroporation, lipofection, and particlegun acceleration.

Treatment: Refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disorder as well as those in which the disorder is to beprevented.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector can include nucleic acidsequences that permit it to replicate in a host cell, such as an originof replication. A vector can also include one or more selectable markergenes and other genetic elements known in the art.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. Hence “comprisingA or B” means including A, or B, or A and B. It is further to beunderstood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including explanations of terms, will control. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

III. Humanized Monoclonal Antibodies that Specifically Bind JEV

Described herein are isolated humanized monoclonal antibodies thatspecifically bind Japanese encephalitis virus (JEV). The humanizedmonoclonal antibodies bind JEV with an affinity constant (K_(d)) ofabout 1.0 nM or less. In some embodiments, the K_(d) is about 0.98 nM,about 0.95 nM or less, about 0.90 nM or less, about 0.85 nM or less,about 0.80 nM or less, about 0.75 nM or less, about 0.72 nM or less,about 0.70 nM or less, about 0.65 nM or less, about 0.60 nM or less, orabout 0.55 nM or less. In other embodiments, the K_(d) is about 0.50 nMor less, about 0.45 nM or less, about 0.40 nM or less, about 0.35 nM orless, about 0.30 nM or less, about 0.28 nM or less, about 0.25 nM, about0.20 nM, about 0.15 nM or about 0.05 nM or less. As used herein, abinding affinity of “about 0.05 nM” includes binding affinities of 0.04nM to 0.06 nM. Similarly, a binding affinity of “about 0.15 nM” includesbinding affinities of 0.1 nM to 0.2 nM.

In some embodiments, the humanized monoclonal antibodies specificallybind JEV envelope protein (ENV). In one embodiment, the human monoclonalantibodies bind the JEV envelope protein of JEV strain SA14-14-2(GenBank Accession No: AF315119). In one non-limiting example, thenucleic acid sequence encoding JEV ENV is

(SEQ ID NO: 9) TTTAATTGTCTGGGAATGGGCAATCGTGACTTCATAGAAGGAGCCAGTGGAGCCACTTGGGTGGACTTGGTGCTAGAAGGAGACAGCTGCTTGACAATCATGGCAAACGACAAACCAACATTGGACGTCCGCATGATTAACATCGAAGCTAGCCAACTTGCTGAGGTCAGAAGTTACTGCTATCATGCTTCAGTCACTGACATCTCGACGGTGGCTCGGTGCCCCACGACTGGAGAAGCCCACAACGAGAAGCGAGCTGATAGTAGCTATGTGTGCAAACAAGGCTTCACTGACCGTGGGTGGGGCAACGGATGTGGATTTTTCGGGAAGGGAAGCATTGACACATGTGCAAAATTCTCCTGCACCAGTAAAGCGATTGGGAGAACAATCCAGCCAGAAAACATCAAATACAAAGTTGGCATTTTTGTGCATGGAACCACCACTTCGGAAAACCATGGGAATTATTCAGCGCAAGTTGGGGCGTCCCAGGCGGCAAAGTTTACAGTAACACCCAATGCTCCTTCGGTAGCCCTCAAACTTGGTGACTACGGAGAAGTCACACTGGACTGTGAGCCAAGGAGTGGACTGAACACTGAAGCGTTTTACGTCATGACCGTGGGGTCAAAGTCATTTCTGGTCCATAGGGAGTGGTTTCATGACCTCGCTCTCCCCTGGACGTCCCCTTCGAGCACAGCGTGGAGAAACAGAGAACTCCTCATGGAATTTGAAGGGGCGCACGCCACAAAACAGTCCGTTGTTGCTCTTGGGTCACAGGAAGGAGGCCTCCATCATGCGTTGGCAGGAGCCATCGTGGTGGAGTACTCAAGCTCAGTGATGTTAACATCAGGCCACCTGAAATGTAGGCTGAAAATGGACAAACTGGCTCTGAAAGGCACAACCTATGGCATGTGTACAGAAAAATTCTCGTTCGCGAAAAATCCGGTGGACACTGGTCACGGAACAGTTGTCATTGAACTCTCCTACTCTGGGAGTGATGGCCCCTGCAAAATTCCGATTGTTTCCGTTGCGAGCCTCAATGACATGACCCCCGTTGGGCGGCTGGTGACAGTGAACCCCTTCGTCGCGACTTCCAGTGCCAACTCAAAGGTGCTGGTCGAGATGGAACCCCCCTTCGGAGACTCCTACATCGTAGTTGGAAGGGGAGACAAGCAGATCAACCACCATTGGCACAAAGCTGGAAGCACGCTGGGCAAGGCCTTTTCAACAACTTTGAAGGGAGCTCAAAGACTGGCAGCGTTGGGCGACACAGCCTGGGACTTTGGCTCTATTGGAGGGGTCTTCAACTCCATAGGAAGAGCCGTTCACCAAGTGTTTGGTGATGCCTTCAGAACACTCTTTGGGGGAATGTCTTGGATCACACAAGGGCTAATGGGTGCCCTACTGCTCTGGATGGGCGTCAACGCACGAGACCGATCAATTGCTTTGGCCTTCTTAGCCACAGGAGGTGTGCTCGTGTTCTTAGCGACCAATGTGCATGCT.

In another non-limiting example, the amino acid sequence of the JEVenvelope protein is

(SEQ ID NO: 10) FNCLGMGNRDFIEGASGATWVDLVLEGDSCLTIMANDKPTLDVRMINIEASQLAEVRSYCYHASVTDISTVARCPTTGEAHNEKRADSSYVCKQGFTDRGWGNGCGFFGKGSIDTCAKFSCTSKAIGRTIQPENIKYKVGIFVHGTTTSENHGNYSAQVGASQAAKFTVTPNAPSVALKLGDYGEVTLDCEPRSGLNTEAFYVMTVGSKSFLVHREWFHDLALPWTSPSSTAWRNRELLMEFEGAHATKQSVVALGSQEGGLHHALAGAIVEYSSSVMLTSGHLKCRLKMDKLALKGTTYGMCTEKFSFAKNPVDTGHGTVVIELSYSGSDGPCKIPIVSVASLNDMTPVGRLVTVNPFVATSSANSKVLVEMEPPFGDSYIVVGRGDKQINHHWHKAGSTLGKAFSTTLKGAQRLAALGDTAWDFGSIGGVFNSIGRAVHQVFGDAFRTLFGGMSWITQGLMGALLLWMGVNARDRSIALAFLATGGVLVFLATNVHA.

The envelope protein is the main functional and antigenic surfacecomponent of the virion. The molecular structure of the ectodomain ofthe envelope protein, which forms a homodimer on the surface of matureviral particles at neutral pH, has been resolved by cryoelectronmicroscopy (Rey et al., Nature 375:291-298, 1995, incorporated byreference herein) and fitted into the electron density map of viralparticles (Kuhn et al., Cell 108:717-725, 2002). The polypeptide chainof the envelope protein folds into three distinct domains: a centraldomain (domain I), a dimerization domain (domain II), and animmunoglobulin-like module domain (domain III). The hinge region ispresent between domains I and II and, upon exposure to acidic pH,undergoes a conformational change (hence the designation “hinge”) thatresults in the formation of envelope protein trimers that are involvedin the fusion of viral and endosomal membranes, after virus uptake byreceptor-mediated endocytosis. The humanized monoclonal antibody canspecifically bind to Lys₁₇₉ within a β-strand in domain I of theenvelope protein. The humanized monoclonal antibody can specificallybind Ile₁₂₆ within the small loop between d and e β-strands in domain IIof the envelope protein. The humanized monoclonal antibody canspecifically bind Gly₃₀₂ within amino acids 302-309 of domain III of theenvelope protein.

In some embodiments, the human monoclonal antibodies include Fabfragments that include chimpanzee CDRs. Further provided arecompositions including the JEV-specific antibodies, nucleic acidsencoding these antibodies, expression vectors including the nucleicacids, and isolated host cells that express the nucleic acids. Alsodescribed are compositions including the provided humanized monoclonalantibodies and a pharmaceutically acceptable carrier. Nucleic acidsencoding these antibodies, expression vectors including these nucleicacids, and isolated host cells that express the nucleic acids also areprovided.

Compositions including the humanized monoclonal antibodies specific forJEV can be used for diagnostic, research and therapeutic purposes. Forexample, the humanized monoclonal antibodies can be used to treat asubject diagnosed with JEV infection, or to prevent the development ofJEV infection in s subject at risk for contracting JEV. The humanizedmonoclonal antibodies also can be used to diagnose JEV infection in asubject. For example, the humanized monoclonal antibodies can becontacted with a sample from the patient, such as a blood sample, todetect JEV in the sample. The antibodies and compositions providedherein can also be used to detect JEV in a subject or to confirm thediagnosis of JEV infection in a patient.

Disclosed herein are humanized monoclonal antibodies that specificallybind JEV. A major limitation in the clinical use of animal monoclonalantibodies, for instance, mouse or sheep monoclonal antibodies is thedevelopment of an anti-mouse or anti-sheep antibody response in thesubjects receiving the treatments. The response can involve allergicreactions and an increased rate of clearance of the administeredantibody from the serum. Various types of modified monoclonal antibodieshave been developed to minimize the antibody response while trying tomaintain the antigen binding affinity of the parent monoclonal antibody.One type of modified monoclonal antibody is a humanized chimera in whichan animal (for instance mouse, sheep or chimpanzee) antigen-bindingvariable region is coupled to a human constant domain (Morrison &Schlom, Important Advances in Oncology, Rosenberg, S. A. (Ed.), 1989). Asecond type of modified monoclonal antibody is the complementaritydetermining region (CDR)-grafted, or humanized, monoclonal antibody(Winter & Harris, Immunol. Today 14:243-246, 1993). Still othermonoclonal antibodies of use include non-human primate antibodies, forexample chimpanzee or macaque antibodies, for which the amino acidsequences are close enough to human sequences that no anti-chimpanzee oranti-macaque antibodies are generated by the subject receiving thetreatment.

In one embodiment, the antibodies bind JEV with an affinity constant(K_(d)) of about 1.0 nM or less. In several embodiments, the humanmonoclonal antibodies bind JEV with a binding affinity of about 0.98 nM,about 0.72 nM or less, about 0.45 nM or less, about 0.35 nM or less,about 0.28 nM or less, or about 0.15 nM or less.

In other embodiments, the antibody is a chimeric antibody, chimpanzeeantibody, or a humanized monoclonal antibody. The antibody can includeone or more CDRs of the heavy chain amino acid sequence set forth as SEQID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In several embodiments, theheavy chain of the antibody comprises: amino acids 31-35 of SEQ ID NO:1, amino acids 50-66 of SEQ ID NO: 1, and/or amino acids 96-107 of SEQID NO: 1. In additional embodiments, the heavy chain of the antibodycomprises amino acids 32-36 of SEQ ID NO: 2, amino acids 50-67 of SEQ IDNO: 2, and/or amino acids 100-113 of SEQ ID NO: 2. In furtherembodiments, the heavy chain of the antibody comprises amino acids 30-37of SEQ ID NO: 3, amino acids 52-67 of SEQ ID NO: 3 and/or amino acids100-112 of SEQ ID NO: 3. Thus, in several examples, the heavy chain ofthe antibody comprises one of (a) amino acids 31-35 of SEQ ID NO: 1,amino acids 50-66 of SEQ ID NO: 1, and amino acids 96-107 of SEQ ID NO:1; (b) amino acids 32-36 of SEQ ID NO: 2, amino acids 50-67 of SEQ IDNO: 2, and amino acids 100-113 of SEQ ID NO: 2; or (c) amino acids 30-37of SEQ ID NO: 3, amino acids 52-67 of SEQ ID NO: 3, and amino acids100-112 of SEQ ID NO: 3. The heavy chain of the antibody can include, orconsist of, one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

In additional embodiments, the antibody can include one or more of theCDRs of the light chain amino acid sequence set forth as SEQ ID NO: 4,SEQ ID NO: 5 or SEQ ID NO: 6. In several embodiments, the light chain ofthe antibody can include amino acids 24-34 of SEQ ID NO: 4, amino acids50-56 of SEQ ID NO: 4 and/or amino acids 89-96 of SEQ ID NO: 4. Infurther embodiments, the light chain of the antibody can include aminoacids 24-34 of SEQ ID NO: 5, amino acids 50-56 of SEQ ID NO: 5; or aminoacids 89-96 of SEQ ID NO: 5. In other embodiments the light chain of theantibody can include amino acids 22-33 of SEQ ID NO: 6, acids 49-55 ofSEQ ID NO 6, and/or amino acids 88-95 of SEQ ID NO: 6.

In several examples, the light chain of the antibody comprises: (a)amino acids 24-34 of SEQ ID NO: 4, amino acids 50-56 of SEQ ID NO: 4,and amino acids 89-96 of SEQ ID NO: 4; (b) amino acids 24-34 of SEQ IDNO: 5. amino acids 50-56 of SEQ ID NO: 5, and amino acids 89-96 of SEQID NO: 5; or (c) amino acids 22-33 of SEQ ID NO: 6, amino acids 49-55 ofSEQ ID NO: 6, and amino acids 88-95 of SEQ ID NO: 6. The heavy chain ofthe antibody can include, or consist of, one of SEQ ID NO: 4, SEQ ID NO:4, or SEQ ID NO: 6.

In one example, the heavy chain of the antibody comprises amino acids31-35, 50-66 and 96-107 of SEQ ID NO: 1, and the light chain of theantibody comprises amino acids 24-34, 50-56 and 89-96 of SEQ ID NO: 4.In another example, the heavy chain of the antibody comprises aminoacids 32-36, 50-67 and 100-113 of SEQ ID NO: 2, and the light chain ofthe antibody comprises amino acids 24-34, 50-56 and 89-96 of SEQ ID NO:5. In a further example, the heavy chain of the antibody comprises aminoacids 30-37, 52-67 and 100-112 of SEQ ID NO: 3, and the light chain ofthe antibody comprises amino acids 22-33, 49-55 and 88-95 of SEQ ID NO:6. In other examples, (a) the heavy chain of the antibody is the aminoacid sequence set forth as SEQ ID NO: 1 and the light chain of theantibody is the amino acid sequence set forth as SEQ ID NO: 4; (b) theheavy chain of the antibody is the amino acid sequence set forth as SEQID NO: 2 and the light chain of the antibody is the amino acid sequenceset forth as SEQ ID NO: 5, or (c) the heavy chain of the antibody is theamino acid sequence set forth as SEQ ID NO: 3 and the light chain of theantibody is the amino acid sequence set forth as SEQ ID NO: 6.

The monoclonal antibody can be of any isotype. The monoclonal antibodycan be, for example, an IgM or an IgG antibody, such as IgG₁ or an IgG₂.The class of an antibody that specifically binds JEV can be switchedwith another. In one aspect, a nucleic acid molecule encoding V_(L) orV_(H) is isolated using methods well-known in the art, such that it doesnot include any nucleic acid sequences encoding the constant region ofthe light or heavy chain, respectively. The nucleic acid moleculeencoding V_(L) or V_(H) is then operatively linked to a nucleic acidsequence encoding a C_(L) or C_(H) from a different class ofimmunoglobulin molecule. This can be achieved using a vector or nucleicacid molecule that includes a C_(L) or C_(H) chain, as known in the art.For example, an antibody that specifically binds JEV that was originallyIgM can be class switched to an IgG. Class switching can be used toconvert one IgG subclass to another, such as from IgG₁ to IgG₂.

Humanized monoclonal antibodies include a human framework region. Thishuman framework region can include the framework regions disclosed inSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, orSEQ ID NO: 6 (these sequences include CDR sequences as well as frameworksequences). In another embodiment, the human framework region caninclude the framework regions disclosed in SEQ ID NO:31, SEQ ID NO: 32,SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36. However,the framework regions can be from another source. Additional examples offramework sequences that can be used include the amino acid frameworksequences of the heavy and light chains disclosed in PCT Publication No.WO 2006/074071 (see, for example, SEQ ID NOs: 1-16), which is hereinincorporated by reference.

Antibody fragments are encompassed by the present disclosure, such asFab, F(ab′)₂, and Fv which include a heavy chain and light chainvariable region and are capable of binding the epitopic determinant onJEV. These antibody fragments retain the ability to selectively bindwith the antigen. These fragments include:

(1) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule, can be produced by digestion of wholeantibody with the enzyme papain to yield an intact light chain and aportion of one heavy chain;

(2) Fab′, the fragment of an antibody molecule can be obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chain; two Fab′ fragmentsare obtained per antibody molecule;

(3) (Fab′)₂, the fragment of the antibody that can be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction; F(ab′)₂ is a dimer of two Fab′ fragments held together by twodisulfide bonds;

(4) Fv, a genetically engineered fragment containing the variable regionof the light chain and the variable region of the heavy chain expressedas two chains; and

(5) Single chain antibody (such as scFv), defined as a geneticallyengineered molecule containing the variable region of the light chain,the variable region of the heavy chain, linked by a suitable polypeptidelinker as a genetically fused single chain molecule.

(6) A dimer of a single chain antibody (scFV₂), defined as a dimer of ascFV. This has also been termed a “miniantibody.”

Methods of making these fragments are known in the art (see for example,Harlow & Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, New York, 1988).

In a further group of embodiments, the antibodies are Fv antibodies,which are typically about 25 kDa and contain a complete antigen-bindingsite with three CDRs per each heavy chain and each light chain. Toproduce these antibodies, the V_(H) and the V_(L) can be expressed fromtwo individual nucleic acid constructs in a host cell. If the V_(H) andthe V_(L) are expressed non-contiguously, the chains of the Fv antibodyare typically held together by noncovalent interactions. However, thesechains tend to dissociate upon dilution, so methods have been developedto crosslink the chains through glutaraldehyde, intermoleculardisulfides, or a peptide linker. Thus, in one example, the Fv can be adisulfide stabilized Fv (dsFv), wherein the heavy chain variable regionand the light chain variable region are chemically linked by disulfidebonds.

In an additional example, the Fv fragments include V_(H) and V_(L)chains connected by a peptide linker. These single-chain antigen bindingproteins (scFv) are prepared by constructing a structural gene includingDNA sequences encoding the V_(H) and V_(L) domains connected by anoligonucleotide. The structural gene is inserted into an expressionvector, which is subsequently introduced into a host cell such as E.coli. The recombinant host cells synthesize a single polypeptide chainwith a linker peptide bridging the two V domains. Methods for producingscFvs are known in the art (see Whitlow et al., Methods: a Companion toMethods in Enzymology, Vol. 2, page 97, 1991; Bird et al., Science242:423, 1988; U.S. Pat. No. 4,946,778; Pack et al., Bio/Technology11:1271, 1993; and Sandhu, supra). Dimers of a single chain antibody(scFV₂), also are contemplated.

Antibody fragments can be prepared by proteolytic hydrolysis of theantibody or by expression in E. coli of DNA encoding the fragment.Antibody fragments can be obtained by pepsin or papain digestion ofwhole antibodies by conventional methods. For example, antibodyfragments can be produced by enzymatic cleavage of antibodies withpepsin to provide a 5S fragment denoted F(ab′)₂. This fragment can befurther cleaved using a thiol reducing agent, and optionally a blockinggroup for the sulfhydryl groups resulting from cleavage of disulfidelinkages, to produce 3.5S Fab′ monovalent fragments. Alternatively, anenzymatic cleavage using pepsin produces two monovalent Fab′ fragmentsand an Fc fragment directly (see U.S. Pat. No. 4,036,945 and U.S. Pat.No. 4,331,647, and references contained therein; Nisonhoff et al., Arch.Biochem. Biophys. 89:230, 1960; Porter, Biochem. J. 73:119, 1959;Edelman et al., Methods in Enzymology, Vol. 1, page 422, Academic Press,1967; and Coligan et al. at sections 2.8.1-2.8.10 and 2.10.1-2.10.4).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques can alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

One of skill will realize that conservative variants of the antibodiescan be produced. Such conservative variants employed in antibodyfragments, such as dsFv fragments or in scFv fragments, will retaincritical amino acid residues necessary for correct folding andstabilizing between the V_(H) and the V_(L) regions, and will retain thecharge characteristics of the residues in order to preserve the low pIand low toxicity of the molecules. Amino acid substitutions (such as atmost one, at most two, at most three, at most four, or at most fiveamino acid substitutions) can be made in the V_(H) and the V_(L) regionsto increase yield. Conservative amino acid substitution tables providingfunctionally similar amino acids are well known to one of ordinary skillin the art. The following six groups are examples of amino acids thatare considered to be conservative substitutions for one another:

-   -   1) Alanine (A), Serine (S), Threonine (T);    -   2) Aspartic acid (D), Glutamic acid (E);    -   3) Asparagine (N), Glutamine (Q);    -   4) Arginine (R), Lysine (K);    -   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and    -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Additional recombinant anti-JEV humanized antibodies can be isolated byscreening additional recombinant combinatorial antibody libraries, suchas a Fab phage display library (see, for example, U.S. Pre-GrantPublication No. 2005/0123900, incorporated herein by reference). In somecases the phage display libraries are prepared using cDNAs of thevariable regions of heavy and light chains prepared from mRNA derivedfrom human lymphocytes. Methodologies for preparing and screening suchlibraries are known in the art. There are commercially available kitsfor generating phage display libraries (for example, the PharmaciaRecombinant Phage Antibody System, catalog no. 27-9400-01; and theStratagene SurfZAP™ phage display kit, catalog no. 240612). There arealso other methods and reagents that can be used in generating andscreening antibody display libraries (see, for example, U.S. Pat. No.5,223,409; PCT Publication No. WO 92/18619; PCT Publication No. WO91/17271; PCT Publication No. WO 92/20791; PCT Publication No. WO92/15679; PCT Publication No. WO 93/01288; PCT Publication No. WO92/01047; PCT Publication No. WO 92/09690; Fuchs et al., Bio/Technology9:1370-1372, 1991; Hay et al., Hum. Antibod. Hybridomas 3:81-85, 1992;Huse et al., Science 246:1275-1281, 1989; McCafferty et al., Nature348:552-554, 1990; Griffiths et al. EMBO J. 12:725-734, 1993).

In one embodiment, to isolate additional human antibodies thatspecifically bind JEV, a human antibody that specifically binds JEV, asdescribed herein, is first used to select human heavy and light chainsequences having similar binding activity toward JEV, such as using theepitope imprinting methods disclosed in PCT Publication No. WO 93/06213.The antibody libraries used in this method are scFv libraries preparedand screened, using methods such as those as described in PCTPublication No. WO 92/01047, McCafferty et al., Nature 348:552-554,1990; and/or Griffiths et al., EMBO J. 12:725-734, 1993 using JEV as theantigen.

Once initial human variable light chain (V_(L)) and variable heavy chain(V_(H)) segments are selected, “mix and match” experiments, in whichdifferent pairs of the initially selected V_(L) and V_(H) segments arescreened for JEV binding, are performed to select V_(L)/V_(H) paircombinations of interest. Additionally, to increase binding affinity ofthe antibody, the V_(L) and V_(H) segments can be randomly mutated, suchas within H-CDR3 region or the L-CDR3 region, in a process analogous tothe in vivo somatic mutation process responsible for affinity maturationof antibodies during a natural immune response. This in vitro affinitymaturation can be accomplished by amplifying V_(H) and V_(L) regionsusing PCR primers complimentary to the H-CDR3 or L-CDR3, respectively.In this process, the primers have been “spiked” with a random mixture ofthe four nucleotide bases at certain positions such that the resultantPCR products encode V_(H) and V_(L) segments into which random mutationshave been introduced into the V_(H) and/or V_(L) CDR3 regions. Theserandomly mutated V_(H) and V_(L) segments can be tested to determine thebinding affinity for JEV.

Following screening and isolation of an antibody that binds JEV from arecombinant immunoglobulin display library, nucleic acid encoding theselected antibody can be recovered from the display package (forexample, from the phage genome) and subcloned into other expressionvectors by standard recombinant DNA techniques, as described herein. Ifdesired, the nucleic acid can be further manipulated to create otherantibody fragments, also as described herein. To express a recombinanthuman antibody isolated by screening of a combinatorial library, the DNAencoding the antibody is cloned into a recombinant expression vector andintroduced into a mammalian host cells, as described herein.

IV. JEV Antibody Polynucleotides and Polypeptides

Nucleic acid molecules (also referred to as polynucleotides) encodingthe polypeptides provided herein (including, but not limited toantibodies) can readily be produced by one of skill in the art, usingthe amino acid sequences provided herein, sequences available in theart, and the genetic code. In addition, one of skill can readilyconstruct a variety of clones containing functionally equivalent nucleicacids, such as nucleic acids which differ in sequence but which encodethe same effector molecule or antibody sequence. Thus, nucleic acidsencoding antibodies, conjugates and fusion proteins are provided herein.In some embodiments, the nucleotide sequence of the heavy chain of theJEV-specific human monoclonal antibody A3 comprises SEQ ID NO: 1, or aportion thereof (such as a portion that encodes one or more CDRs). Insome embodiments, the amino acid sequence of the light chain of theJEV-specific human monoclonal antibody A3 comprises SEQ ID NO: 4, or aportion thereof (such as a portion that encodes one or more CDRs).

In one embodiment, the nucleotide sequence of the heavy chain of theJEV-specific human monoclonal antibody B2 comprises SEQ ID NO: 2, or aportion thereof (such as a portion that encodes one or more CDRs). Inone embodiment, the amino acid sequence of the light chain of theJEV-specific human monoclonal antibody B2 comprises SEQ ID NO: 5, or aportion thereof (such as a portion that encodes one or more CDRs).

In yet another embodiment, the nucleotide sequence of the heavy chain ofthe JEV-specific human monoclonal antibody E3 comprises SEQ ID NO: 3, ora portion thereof (such as a portion that encodes one or more CDRs). Inanother embodiment, the amino acid sequence of the light chain of theJEV-specific human monoclonal antibody E3 comprises SEQ ID NO: 6, or aportion thereof (such as a portion that encodes one or more CDRs).

Nucleic acid sequences encoding the humanized antibodies thatspecifically bind JEV can be prepared by any suitable method including,for example, cloning of appropriate sequences or by direct chemicalsynthesis by methods such as the phosphotriester method of Narang etal., Meth. Enzymol. 68:90-99, 1979; the phosphodiester method of Brownet al., Meth. Enzymol. 68:109-151, 1979; the diethylphosphoramiditemethod of Beaucage et al., Tetra. Lett. 22:1859-1862, 1981; the solidphase phosphoramidite triester method described by Beaucage & Caruthers,Tetra. Letts. 22(20):1859-1862, 1981, for example, using an automatedsynthesizer as described in, for example, Needham-VanDevanter et al.,Nucl. Acids Res. 12:6159-6168, 1984; and, the solid support method ofU.S. Pat. No. 4,458,066. Chemical synthesis produces a single strandedoligonucleotide. This can be converted into double stranded DNA byhybridization with a complementary sequence or by polymerization with aDNA polymerase using the single strand as a template. One of skill wouldrecognize that while chemical synthesis of DNA is generally limited tosequences of about 100 bases, longer sequences can be obtained by theligation of shorter sequences.

Exemplary nucleic acids encoding humanized antibodies that specificallybind JEV can be prepared by cloning techniques. Examples of appropriatecloning and sequencing techniques, and instructions sufficient to directpersons of skill through many cloning exercises are found in Sambrook etal., supra, Berger and Kimmel (eds.), supra, and Ausubel, supra. Productinformation from manufacturers of biological reagents and experimentalequipment also provide useful information. Such manufacturers includethe SIGMA Chemical Company (Saint Louis, Mo.), R&D Systems (Minneapolis,Minn.), Pharmacia Amersham (Piscataway, N.J.), CLONTECH Laboratories,Inc. (Palo Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company(Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL Life Technologies,Inc. (Gaithersburg, Md.), Fluka Chemica-Biochemika Analytika (FlukaChemie AG, Buchs, Switzerland), Invitrogen (Carlsbad, Calif.), andApplied Biosystems (Foster City, Calif.), as well as many othercommercial sources known to one of skill

Nucleic acids encoding anti-JEV antibodies can be modified to form theantibodies of the present disclosure. Modification by site-directedmutagenesis is well known in the art. Nucleic acids also can be preparedby amplification methods. Amplification methods include polymerase chainreaction (PCR), the ligase chain reaction (LCR), the transcription-basedamplification system (TAS), the self-sustained sequence replicationsystem (3SR). A wide variety of cloning methods, host cells, and invitro amplification methodologies are well known to persons of skill.

In one embodiment, antibodies are prepared by inserting the cDNA whichencodes a humanized JEV-specific monoclonal antibody into a vector. Inone embodiment, cDNA encoding a label or enzyme is ligated to anantibody so that the label or enzyme is located at the carboxyl terminusof the antibody. In another embodiment, the label or enzyme is locatedat the amino terminus of the antibody. In another example, cDNA encodingthe label or enzyme is ligated to a heavy chain variable region of anantibody, so that the label or enzyme is located at the carboxylterminus of the heavy chain variable region. The heavy chain-variableregion can subsequently be ligated to a light chain variable region ofthe antibody using disulfide bonds. In a yet another example, cDNAencoding a label or enzyme is ligated to a light chain variable regionof an antibody, so that the label or enzyme is located at the carboxylterminus of the light chain variable region. The light chain-variableregion can subsequently be ligated to a heavy chain variable region ofthe antibody using disulfide bonds.

Once the nucleic acids encoding the anti-JEV antibody are isolated andcloned, the desired protein can be expressed in a recombinantlyengineered cell such as bacteria, yeast, insect and mammalian cells. Itis expected that those of skill in the art are knowledgeable in thenumerous expression systems available for expression of proteinsincluding E. coli, other bacterial hosts, yeast, and various highereukaryotic cells such as the COS, CHO, HeLa and myeloma cell lines.

One or more DNA sequences encoding the antibody or fragment thereof canbe expressed in vitro by DNA transfer into a suitable host cell. Thecell can be prokaryotic or eukaryotic. The term also includes anyprogeny of the subject host cell. It is understood that all progeny cannot be identical to the parental cell since there can be mutations thatoccur during replication. Methods of stable transfer, meaning that theforeign DNA is continuously maintained in the host, are known in theart. Hybridomas expressing the antibodies of interest are alsoencompassed by this disclosure.

The expression of nucleic acids encoding the isolated proteins describedherein can be achieved by operably linking the DNA or cDNA to a promoter(which is either constitutive or inducible), followed by incorporationinto an expression cassette. The cassettes can be suitable forreplication and integration in either prokaryotes or eukaryotes. Typicalexpression cassettes contain specific sequences useful for regulation ofthe expression of the DNA encoding the protein. For example, theexpression cassettes can include appropriate promoters, enhancers,transcription and translation terminators, initiation sequences, a startcodon (for instance, ATG) in front of a protein-encoding gene, splicingsignal for introns, maintenance of the correct reading frame of thatgene to permit proper translation of mRNA, and stop codons.

To obtain high level expression of a cloned gene, it is desirable toconstruct expression cassettes which contain, at the minimum, a strongpromoter to direct transcription, a ribosome binding site fortranslational initiation, and a transcription/translation terminator.For E. coli, this includes a promoter such as the T7, trp, lac, orlambda promoters, a ribosome binding site, and preferably atranscription termination signal. For eukaryotic cells, the controlsequences can include a promoter and/or an enhancer derived from, forexample, an immunoglobulin gene, SV40 or cytomegalovirus, and apolyadenylation sequence, and can further include splice donor andacceptor sequences. The cassettes can be transferred into the chosenhost cell by well-known methods such as transformation orelectroporation for E. coli and calcium phosphate treatment,electroporation or lipofection for mammalian cells. Cells transformed bythe cassettes can be selected by resistance to antibiotics conferred bygenes contained in the cassettes, such as the amp, gpt, neo and hyggenes.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate coprecipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors can be used. Eukaryotic cells can also becotransformed with polynucleotide sequences encoding the antibody,labeled antibody, or functional fragment thereof, and a second foreignDNA molecule encoding a selectable phenotype, such as the herpes simplexthymidine kinase gene. Another method is to use a eukaryotic viralvector, such as simian virus 40 (SV40) or bovine papilloma virus, totransiently infect or transform eukaryotic cells and express the protein(see for example, Eukaryotic Viral Vectors, Cold Spring HarborLaboratory, Gluzman ed., 1982). One of skill in the art can readily usean expression systems such as plasmids and vectors of use in producingproteins in cells including higher eukaryotic cells such as the COS,CHO, HeLa and myeloma cell lines.

Modifications can be made to a nucleic acid encoding a polypeptidedescribed herein (for instance, a humanized JEV-specific monoclonalantibody) without diminishing its biological activity. Somemodifications can be made to facilitate the cloning, expression, orincorporation of the targeting molecule into a fusion protein. Suchmodifications are well known to those of skill in the art and include,for example, termination codons, a methionine added at the aminoterminus to provide an initiation, site, additional amino acids placedon either terminus to create conveniently located restriction sites, oradditional amino acids (such as poly His) to aid in purification steps.Modification can also be used to construct an Fc deletion (see below).In addition to recombinant methods, the antibodies of the presentdisclosure can also be constructed in whole or in part using standardpeptide synthesis well known in the art.

Once expressed, the recombinant antibodies can be purified according tostandard procedures of the art, including ammonium sulfateprecipitation, affinity columns, column chromatography, and the like(see, generally, R. Scopes, Protein Purification, Springer-Verlag, N.Y.,1982). The antibodies, immunoconjugates and effector molecules need notbe 100% pure. Once purified, partially or to homogeneity as desired, ifto be used therapeutically, the polypeptides should be substantiallyfree of endotoxin.

Methods for expression of single chain antibodies and/or refolding to anappropriate active form, including single chain antibodies, frombacteria such as E. coli have been described and are well-known and areapplicable to the antibodies disclosed herein. See, Buchner et al.,(1992) Anal. Biochem. 205:263-270; Pluckthun, (1991) Biotechnology9:545; Huse et al., (1989) Science 246:1275; and Ward et al., (1989)Nature 341:544.

Often, functional heterologous proteins from E. coli or other bacteriaare isolated from inclusion bodies and require solubilization usingstrong denaturants, and subsequent refolding. During the solubilizationstep, as is well known in the art, a reducing agent must be present toseparate disulfide bonds. An exemplary buffer with a reducing agent is:0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol).Reoxidation of the disulfide bonds can occur in the presence of lowmolecular weight thiol reagents in reduced and oxidized form, asdescribed in Saxena et al., Biochemistry 9: 5015-5021, 1970,incorporated by reference herein, and especially as described by Buchneret al., supra.

Renaturation is typically accomplished by dilution (for example,100-fold) of the denatured and reduced protein into refolding buffer. Anexemplary buffer is 0.1 M Tris, pH 8.0, 0.5 M L-arginine, 8 mM oxidizedglutathione, and 2 mM EDTA.

As a modification to the two chain antibody purification protocol, theheavy and light chain regions are separately solubilized and reduced andthen combined in the refolding solution. An exemplary yield is obtainedwhen these two proteins are mixed in a molar ratio such that a 5-foldmolar excess of one protein over the other is not exceeded. Excessoxidized glutathione or other oxidizing low molecular weight compoundscan be added to the refolding solution after the redox-shuffling iscompleted.

In addition to recombinant methods, the antibodies, labeled antibodiesand functional fragments thereof that are disclosed herein can also beconstructed in whole or in part using standard peptide synthesis. Solidphase synthesis of the polypeptides of less than about 50 amino acids inlength can be accomplished by attaching the C-terminal amino acid of thesequence to an insoluble support followed by sequential addition of theremaining amino acids in the sequence. Techniques for solid phasesynthesis are described by Barany & Merrifield, The Peptides: Analysis,Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, PartA. pp. 3-284; Merrifield et al., (1963) J. Am. Chem. Soc. 85:2149-2156;and Stewart et al., (1984) Solid Phase Peptide Synthesis, 2nd ed.,Pierce Chem. Co., Rockford, Ill. Proteins of greater length can besynthesized by condensation of the amino and carboxyl termini of shorterfragments. Methods of forming peptide bonds by activation of a carboxylterminal end (such as by the use of the coupling reagentN,N′-dicylohexylcarbodimide) are well known in the art.

V. Fc Deletion Mutants with Reduced Fcγ Receptor Binding Affinity

Also disclosed herein are deletion mutants of the JEV antibodies thathave a deletion of about nine amino acids (from about position 231 toabout position 239) at the N-terminus of the C_(H)2 domain in the Fcregion. By a deletion of “about nine” amino acids are meant a deletionof at least one amino acid from about position 231 to about position 239at the N-terminus of the C_(H)2 domain in the Fc region. In someembodiments, deletion of “about nine” amino acids refers to a deletionof about 4 amino acids having the sequence LLGG (SEQ ID NO: 12), and nomore than 10 amino acids. In another embodiment, a deletion of “aboutnine” amino acids is meant a deletion of one, two, three, four, five,six, seven, eight, nine or ten amino acids from about position 231 toabout position 239 at the N-terminus of the C_(H)2 domain in the Fcregion. The Fcγ receptor class I binding site in human IgG has beenidentified as the sequence spanning residues 234-237 (LLGG; Chappel etal. Proc Natl Acad Sci USA 88:9036-9040). For example, such deletions(Table 1) remove the following amino acids:

TABLE 1 Deleted Sequences Number of Amino Acids Deleted Deleted SequenceSEQ ID NO: 4 LLGG 12 5        ELLGG 13         LLGGP 14 6       PELLGG15        ELLGGP 16         LLGGPS 17 7      APELLGG 18       PELLGGP 19       ELLGGPS 20         LLGGPSV 21 8     PAPELLGG 22      APELLGGP 23      PELLGGPS 24        ELLGGPSV 25 9     PAPELLGGP 26      APELLGGPS27       PELLGGPSV 28 10     PAPELLGGPS 29      APELLGGPSV 30

The “parent”, “starting” or “nonvariant” polypeptide is prepared usingtechniques available in the art for generating polypeptides including anFc region. In some embodiments, the parent polypeptide is a humanizedmonoclonal antibody that specifically binds JEV. A variant Fc region canbe generated and this “variant Fc region” then can be fused to aheterologous antibody variable domain of the humanized monoclonalantibody that specifically binds JEV and/or JEV envelope protein.

The parent polypeptide includes an Fc region. Generally the Fc region ofthe parent polypeptide will include a native sequence Fc region, andpreferably a human native sequence Fc region. However, the Fc region ofthe parent polypeptide can have one or more pre-existing amino acidsequence alterations or modifications from a native sequence Fc region.In a further embodiment the parent polypeptide Fc region is “conceptual”and, while it does not physically exist, the antibody engineer candecide upon a desired variant Fc region amino acid sequence and generatea polypeptide including that sequence or a DNA encoding the desiredvariant Fc region amino acid sequence. In some embodiments, however, anucleic acid encoding an Fc region of a parent polypeptide is availableand this nucleic acid sequence is altered to generate a variant nucleicacid sequence encoding the Fc region variant.

DNA encoding an amino acid sequence variant of the starting polypeptideis prepared by a variety of methods known in the art. These methodsinclude, but are not limited to, preparation by site-directed (oroligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassettemutagenesis of an earlier prepared DNA encoding the polypeptide.Site-directed mutagenesis is a preferred method for preparingsubstitution variants. This technique is well known in the art (see, forinstance, Kunkel et al. 1987 Proc Natl Acad Sci USA 82:488). Briefly, incarrying out site-directed mutagenesis of DNA, the starting DNA isaltered by first hybridizing an oligonucleotide encoding the desiredmutation to a single strand of such starting DNA. After hybridization, aDNA polymerase is used to synthesize an entire second strand, using thehybridized oligonucleotide as a primer, and using the single strand ofthe starting DNA as a template. Thus, the oligonucleotide encoding thedesired mutation is incorporated in the resulting double-stranded DNA.

PCR mutagenesis is also suitable for making amino acid sequence variantsof the starting polypeptide. See PCR Protocols: A guide to methods andapplications, Michael A. Innis, chapter by Higuchi, pp. 177-183(Academic Press, 1990). Briefly, when small amounts of template DNA areused as starting material in a PCR, primers that differ slightly insequence from the corresponding region in a template DNA can be used togenerate relatively large quantities of a specific DNA fragment thatdiffers from the template sequence only at the positions where theprimers differ from the template.

Another method for preparing variants, cassette mutagenesis, is based onthe technique described by Wells et al. 1985 Gene 34:315-323. Thestarting material is the plasmid (or other vector) including thestarting polypeptide DNA to be mutated. The codon(s) in the starting DNAto be mutated are identified. There must be a unique restrictionendonuclease site on each side of the identified mutation site(s). If nosuch restriction sites exist, they can be generated using theabove-described oligonucleotide-mediated mutagenesis method to introducethem at appropriate locations in the starting polypeptide DNA. Theplasmid DNA is cut at these sites to linearize it. A double-strandedoligonucleotide encoding the sequence of the DNA between the restrictionsites but containing the desired mutation(s) is synthesized usingstandard procedures, wherein the two strands of the oligonucleotide aresynthesized separately and then hybridized together using standardtechniques. This double-stranded oligonucleotide is referred to as thecassette. This cassette is designed to have 5′ and 3′ ends that arecompatible with the ends of the linearized plasmid, such that it can bedirectly ligated to the plasmid. This plasmid now contains the mutatedDNA sequence. Alternatively, or additionally, the desired amino acidsequence encoding a polypeptide variant can be determined, and a nucleicacid sequence encoding such amino acid sequence variant can be generatedsynthetically.

The amino acid sequence of the parent polypeptide, specifically thehumanized monoclonal antibody that specifically binds JEV is modified inorder to generate a variant Fc region with altered Fc receptor bindingaffinity or activity in vitro and/or in vivo. For example, about nineamino acids (from about position 231 to about position 239) can bedeleted at the N-terminus of the C_(H)2 domain in the Fc region. The Fcregion herein including one or more amino acid deletions can retain atleast about 80%, such as at least about 90%, such as at least about 95%,of the parent Fc region or of a native sequence human Fc region. In someembodiments, the parent polypeptide Fc region is a human Fc region, forinstance, a native sequence human Fc region human IgG1 (A and non-Aallotypes), IgG2, IgG3 or IgG4 Fc region.

The humanized monoclonal antibodies prepared as described above can besubjected to further modifications, oftentimes depending on the intendeduse of the polypeptide. Such modifications can involve furtheralteration of the amino acid sequence (substitution, insertion and/ordeletion of amino acid residues), fusion to heterologous polypeptide(s)and/or covalent modifications. Such “further modifications” can be madeprior to, simultaneously with, or following, the amino acidmodification(s) disclosed above that result in an alteration of Fcreceptor binding. In one embodiment, one can combine the Fc regionmodification herein with another Fc region modification. Alternativelyor additionally, it can be useful to combine the above amino acidmodifications with one or more further amino acid modifications thatalter FcRn binding and/or half-life of the antibody.

With respect to further amino acid sequence alterations, any cysteineresidue not involved in maintaining the proper conformation of thehumanized antibody also can be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcross linking. The humanized monoclonal antibody that specifically bindsJEV can be subjected to one or more assays to evaluate any change inbiological activity compared to the starting polypeptide. Preferably thehumanized antibody monoclonal antibody that specifically binds JEVretains the ability to bind antigen compared to the nonvariantpolypeptide, for instance, the binding capability is no worse than about20 fold, for instance, no worse than about 5 fold of that of thenonvariant polypeptide. The binding capability of the humanized antibodycan be determined using techniques such as fluorescence activated cellsorting (FACS) analysis or radioimmunoprecipitation (RIA), for example.The ability of the humanized antibody to bind an FcR can be evaluated.Where the FcR is a high affinity Fc receptor, such as FcγRI, FcRn orFcγRIIIA-V158, binding can be measured by titrating monomeric humanizedantibody and measuring bound humanized antibody using an antibody thatspecifically binds to the humanized antibody in a standard ELISA format.

VI. Labeled Humanized Antibodies

The human monoclonal antibodies specific for JEV described herein can beconjugated to a therapeutic agent. Immunoconjugates include, but are notlimited to, molecules in which there is a covalent linkage of atherapeutic agent to an antibody. A therapeutic agent is an agent with aparticular biological activity directed against a particular targetmolecule or a cell bearing a target molecule. One of skill in the artwill appreciate that therapeutic agents can include various drugs suchas vinblastine, daunomycin and the like, cytotoxins such as native ormodified Pseudomonas exotoxin or Diphtheria toxin, encapsulating agents(such as liposomes) which themselves contain pharmacologicalcompositions, radioactive agents such as ¹²⁵I, ³²P, ¹⁴C, ³H and ³⁵S andother labels, target moieties and ligands.

The choice of a particular therapeutic agent depends on the particulartarget molecule or cell, and the desired biological effect. Thus, forexample, the therapeutic agent can be a cytotoxin that is used to bringabout the death of a particular target cell. Conversely, where it isdesired to invoke a non-lethal biological response, the therapeuticagent can be conjugated to a non-lethal pharmacological agent or aliposome containing a non-lethal pharmacological agent.

With the therapeutic agents and antibodies described herein, one ofskill can readily construct a variety of clones containing functionallyequivalent nucleic acids, such as nucleic acids which differ in sequencebut which encode the same EM or antibody sequence. Thus, the presentinvention provides nucleic acids encoding antibodies and conjugates andfusion proteins thereof.

Effector molecules can be linked to an antibody of interest using anynumber of means known to those of skill in the art. Both covalent andnoncovalent attachment means may be used. The procedure for attaching aneffector molecule to an antibody varies according to the chemicalstructure of the effector. Polypeptides typically contain a variety offunctional groups; such as carboxylic acid (COOH), free amine (—NH₂) orsulfhydryl (—SH) groups, which are available for reaction with asuitable functional group on an antibody to result in the binding of theeffector molecule. Alternatively, the antibody is derivatized to exposeor attach additional reactive functional groups. The derivatization mayinvolve attachment of any of a number of linker molecules such as thoseavailable from Pierce Chemical Company, Rockford, Ill. The linker can beany molecule used to join the antibody to the effector molecule. Thelinker is capable of forming covalent bonds to both the antibody and tothe effector molecule. Suitable linkers are well known to those of skillin the art and include, but are not limited to, straight orbranched-chain carbon linkers, heterocyclic carbon linkers, or peptidelinkers. Where the antibody and the effector molecule are polypeptides,the linkers may be joined to the constituent amino acids through theirside groups (such as through a disulfide linkage to cysteine) or to thealpha carbon amino and carboxyl groups of the terminal amino acids.

In some circumstances, it is desirable to free the effector moleculefrom the antibody when the immunoconjugate has reached its target site.Therefore, in these circumstances, immunoconjugates will compriselinkages that are cleavable in the vicinity of the target site. Cleavageof the linker to release the effector molecule from the antibody may beprompted by enzymatic activity or conditions to which theimmunoconjugate is subjected either inside the target cell or in thevicinity of the target site.

In view of the large number of methods that have been reported forattaching a variety of radiodiagnostic compounds, radiotherapeuticcompounds, label (such as enzymes or fluorescent molecules) drugs,toxins, and other agents to antibodies one skilled in the art will beable to determine a suitable method for attaching a given agent to anantibody or other polypeptide.

The humanized monoclonal antibodies or antibody fragments thatspecifically bind JEV disclosed herein can be derivatized or linked toanother molecule (such as another peptide or protein). In general, theantibodies or portion thereof is derivatized such that the binding toJEV is not affected adversely by the derivatization or labeling. Forexample, the antibody can be functionally linked (by chemical coupling,genetic fusion, noncovalent association or otherwise) to one or moreother molecular entities, such as another antibody (for example, abispecific antibody or a diabody), a detection agent, a pharmaceuticalagent, and/or a protein or peptide that can mediate associate of theantibody or antibody portion with another molecule (such as astreptavidin core region or a polyhistidine tag).

One type of derivatized antibody is produced by cross-linking two ormore antibodies (of the same type or of different types, such as tocreate bispecific antibodies). Suitable crosslinkers include those thatare heterobifunctional, having two distinctly reactive groups separatedby an appropriate spacer (such asm-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (suchas disuccinimidyl suberate). Such linkers are available from PierceChemical Company, Rockford, Ill.

A humanized antibody that specifically binds JEV can be labeled with adetectable moiety. Useful detection agents include fluorescentcompounds, including fluorescein, fluorescein isothiocyanate, rhodamine,5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin, lanthanidephosphors and the like. Bioluminescent markers are also of use, such asluciferase, Green fluorescent protein (GFP), Yellow fluorescent protein(YFP). An antibody can also be labeled with enzymes that are useful fordetection, such as horseradish peroxidase, β-galactosidase, luciferase,alkaline phosphatase, glucose oxidase and the like. When an antibody islabeled with a detectable enzyme, it can be detected by addingadditional reagents that the enzyme uses to produce a reaction productthat can be discerned. For example, when the agent horseradishperoxidase is present the addition of hydrogen peroxide anddiaminobenzidine leads to a colored reaction product, which is visuallydetectable. An antibody also can be labeled with biotin, and detectedthrough indirect measurement of avidin or streptavidin binding. Itshould be noted that the avidin itself can be labeled with an enzyme ora fluorescent label.

An antibody can be labeled with a magnetic agent, such as gadolinium.Antibodies can also be labeled with lanthanides (such as europium anddysprosium), and manganese. Paramagnetic particles such assuperparamagnetic iron oxide are also of use as labels. An antibody canalso be labeled with a predetermined polypeptide epitopes recognized bya secondary reporter (such as leucine zipper pair sequences, bindingsites for secondary antibodies, metal binding domains, epitope tags). Insome embodiments, labels are attached by spacer arms of various lengthsto reduce potential steric hindrance.

An antibody also can be labeled with a radiolabeled amino acid. Theradiolabel can be used for both diagnostic and therapeutic purposes. Forinstance, the radiolabel can be used to detect JEV by x-ray, emissionspectra, or other diagnostic techniques. Examples of labels forpolypeptides include, but are not limited to, the followingradioisotopes or radionucleotides: ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In,¹²⁵I, ¹³¹I.

An antibody can also be derivatized with a chemical group such aspolyethylene glycol, a methyl or ethyl group, or a carbohydrate group.These groups can be useful to improve the biological characteristics ofthe antibody, such as to increase serum half-life or to increase tissuebinding.

Toxins can be employed with the JEV-specific human monoclonal antibodiesdescribed herein to produce immunotoxins. Exemplary toxins includericin, abrin, diphtheria toxin and subunits thereof, as well asbotulinum toxins A through F. These toxins are readily available fromcommercial sources (for example, Sigma Chemical Company, St. Louis,Mo.). Contemplated toxins also include variants of the toxins describedherein (see, for example, see, U.S. Pat. Nos. 5,079,163 and 4,689,401).

The JEV-specific antibodies described herein can also be used to targetany number of different diagnostic or therapeutic compounds to cellsexpressing JEV on their surface. Thus, an antibody of the presentdisclosure can be attached directly or via a linker to a drug that is tobe delivered directly to cells expressing cell-surface JEV. This can bedone for therapeutic or research purposes. Therapeutic agents includesuch compounds as nucleic acids, proteins, peptides, amino acids orderivatives, glycoproteins, radioisotopes, lipids, carbohydrates, orrecombinant viruses. Nucleic acid therapeutic and diagnostic moietiesinclude antisense nucleic acids, derivatized oligonucleotides forcovalent cross-linking with single or duplex DNA, and triplex formingoligonucleotides.

Alternatively, the molecule linked to an anti-JEV antibody can be anencapsulation system, such as a liposome or micelle that contains atherapeutic composition such as a drug, a nucleic acid (for example, anantisense nucleic acid), or another therapeutic moiety that ispreferably shielded from direct exposure to the circulatory system.Means of preparing liposomes attached to antibodies are well known tothose of skill in the art (see, for example, U.S. Pat. No. 4,957,735;Connor et al., Pharm. Ther. 28:341-365, 1985).

Antibodies described herein can also be covalently or non-covalentlylinked to a detectable label. Detectable labels suitable for such useinclude any composition detectable by spectroscopic, photochemical,biochemical, immunochemical, electrical, optical or chemical means.Useful labels include magnetic beads, fluorescent dyes (for example,fluorescein isothiocyanate, Texas red, rhodamine, green fluorescentprotein, and the like), radiolabels (for example, ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P), enzymes (such as horseradish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), and colorimetric labels such ascolloidal gold or colored glass or plastic (such as polystyrene,polypropylene, latex, and the like) beads.

Means of detecting such labels are well known to those of skill in theart. Thus, for example, radiolabels can be detected using photographicfilm or scintillation counters, fluorescent markers can be detectedusing a photodetector to detect emitted illumination. Enzymatic labelsare typically detected by providing the enzyme with a substrate anddetecting the reaction product produced by the action of the enzyme onthe substrate, and colorimetric labels are detected by simplyvisualizing the colored label.

VII. Pharmaceutical Compositions

Compositions are provided that include one or more of the antibodiesthat specifically bind JEV that are disclosed herein in a carrier. Thecompositions can be prepared in unit dosage forms for administration toa subject. The amount and timing of administration are at the discretionof the treating physician to achieve the desired purposes. The antibodycan be formulated for systemic or local (such as inhalational)administration. In one example, the antibody that specifically binds JEVis formulated for parenteral administration, such as intravenous orintramuscular administration.

The compositions for administration can include a solution of theantibody that specifically binds JEV dissolved in a pharmaceuticallyacceptable carrier, such as an aqueous carrier. A variety of aqueouscarriers can be used, for example, buffered saline and the like. Thesesolutions are sterile and generally free of undesirable matter. Thesecompositions can be sterilized by conventional, well known sterilizationtechniques. The compositions can contain pharmaceutically acceptableauxiliary substances as required to approximate physiological conditionssuch as pH adjusting and buffering agents, toxicity adjusting agents andthe like, for example, sodium acetate, sodium chloride, potassiumchloride, calcium chloride, sodium lactate and the like. Theconcentration of antibody in these formulations can vary widely, andwill be selected primarily based on fluid volumes, viscosities, bodyweight and the like in accordance with the particular mode ofadministration selected and the subject's needs.

A typical pharmaceutical composition for intravenous or intramuscularadministration includes about 100 mg to about 5 g of antibody persubject per day. Dosages from 200 mg up to about 15 g of antibody persubject per day can be used, particularly if the agent is administeredto a secluded site and not into the circulatory or lymph system, such asinto a body cavity or into a lumen of an organ. Actual methods forpreparing administrable compositions will be known or apparent to thoseskilled in the art and are described in more detail in such publicationsas Remington's Pharmaceutical Science, 19th ed., Mack PublishingCompany, Easton, Pa. (1995).

Antibodies can be provided in lyophilized form and rehydrated withsterile water before administration, although they are also provided insterile solutions of known concentration. The antibody solution is thenadded to an infusion bag containing 0.9% sodium chloride, USP, andtypically administered at a dosage of from 5 to 300 mg/kg of bodyweight. Considerable experience is available in the art in theadministration of antibody drugs, which have been marketed in the U.S.since the approval of RITUXAN® in 1997. Antibodies can be administeredby slow infusion, rather than in an intravenous push or bolus. In oneexample, a higher loading dose is administered, with subsequent,maintenance doses being administered at a lower level. For example, aninitial loading dose of 50 mg/kg can be infused over a period of some 90minutes, followed by weekly maintenance doses for 4-8 weeks of 5 mg/kginfused over a 30 minute period if the previous dose was well tolerated.

Single or multiple administrations of the compositions are administereddepending on the dosage and frequency as required and tolerated by thepatient. In any event, the composition should provide a sufficientquantity of at least one of the antibodies disclosed herein toeffectively treat the patient. The dosage can be administered once butmay be applied periodically until either a therapeutic result isachieved or until side effects warrant discontinuation of therapy. Inone example, a dose of the antibody is infused for thirty minutes everyother day. In this example, about one to about ten doses can beadministered, such as three or six doses can be administered every otherday. In a further example, a continuous infusion is administered forabout five to about ten days. The subject can be treated at regularintervals, such as monthly, until a desired therapeutic result isachieved. Generally, the dose is sufficient to treat or amelioratesymptoms or signs of disease without producing unacceptable toxicity tothe patient.

Controlled release parenteral formulations can be made as implants, oilyinjections, or as particulate systems. For a broad overview of proteindelivery systems see, Banga (1995) Therapeutic Peptides and Proteins:Formulation, Processing, and Delivery Systems, Technomic PublishingCompany, Inc., Lancaster, Pa. Particulate systems include microspheres,microparticles, microcapsules, nanocapsules, nanospheres, andnanoparticles. Microcapsules contain the therapeutic protein, such as acytotoxin or a drug, as a central core. In microspheres the therapeuticis dispersed throughout the particle. Particles, microspheres, andmicrocapsules smaller than about 1 μm are generally referred to asnanoparticles, nanospheres, and nanocapsules, respectively. Capillarieshave a diameter of approximately 5 μm so that only nanoparticles areadministered intravenously. Microparticles are typically around 100 μmin diameter and are administered subcutaneously or intramuscularly. See,for example, Kreuter (1994) Colloidal Drug Delivery Systems, J. Kreuter,ed., Marcel Dekker, Inc., New York, N.Y., pp. 219-342; and Tice & Tabibi(1992) Treatise on Controlled Drug Delivery, A. Kydonieus, ed., MarcelDekker, Inc. New York, N.Y., pp. 315-339.

Polymers can be used for ion-controlled release of the antibodycompositions disclosed herein. Various degradable and nondegradablepolymeric matrices for use in controlled drug delivery are known in theart (Langer (1993) Accounts Chem. Res. 26:537-542). For example, theblock copolymer, polaxamer 407, exists as a viscous yet mobile liquid atlow temperatures but forms a semisolid gel at body temperature. It hasbeen shown to be an effective vehicle for formulation and sustaineddelivery of recombinant interleukin-2 and urease (Johnston et al.,(1992) Pharm. Res. 9:425-434; and Pec et al., (1990) J. Parent. Sci.Tech. 44(2):58-65). Alternatively, hydroxyapatite has been used as amicrocarrier for controlled release of proteins (Ijntema et al., Int. J.Pharm. 112:215-224, 1994). In yet another aspect, liposomes are used forcontrolled release as well as drug targeting of the lipid-capsulateddrug (Betageri et al., (1993) Liposome Drug Delivery Systems, TechnomicPublishing Co., Inc., Lancaster, Pa.). Numerous additional systems forcontrolled delivery of therapeutic proteins are known (see U.S. Pat.Nos. 5,055,303; 5,188,837; 4,235,871; 4,501,728; 4,837,028; 4,957,735;5,019,369; 5,055,303; 5,514,670; 5,413,797; 5,268,164; 5,004,697;4,902,505; 5,506,206; 5,271,961; 5,254,342 and 5,534,496).

VIII. Methods of Treatment and Prophylaxis

The humanized antibodies disclosed herein can be used for theprophylaxis or treatment of a mammal, for instance, a human subject whohas been diagnosed with JEV infection, or a person at risk for exposureto JEV. In some embodiments, the antibody that specifically binds JEV isadministered to treat or inhibit the development of JEV infection. Inthese applications, a therapeutically effective amount of an antibody isadministered to a subject in an amount sufficient to treat the JEVinfection. In other embodiments, the antibody is administered to asubject at risk for exposure to JEV in order to inhibit the developmentof JEV infection. Suitable subjects can include those diagnosed with aJEV infection, a subject suspected of having contracted JEV, or asubject at risk for exposure to JEV, for instance a health care workeror other subject who lives in or travels to an area where JEV isendemic. Any active form of the antibody can be administered, includingFab and F(ab′)2 fragments.

The humanized antibody is administered by any suitable means, includingparenteral, subcutaneous, intraperitoneal, intrapulmonary, andintranasal. Parenteral infusions include intramuscular, intravenous,intracerebral, intraarterial, intraperitoneal, or subcutaneousadministration. In addition, the humanized antibody is suitablyadministered by pulse infusion, particularly with declining doses of thehumanized antibody. In some embodiments, the dosing is given byinjections, most preferably intravenous or intramuscular injections,depending in part on whether the administration is brief or chronic.

For the prevention or treatment of disease, such as JEV infection, theappropriate dosage of humanized antibody will depend on the severity andcourse of the disease, whether the humanized antibody is administeredfor preventive or therapeutic purposes, previous prophylaxis andtherapy, the subject's clinical history and response to the humanizedantibody, and the discretion of the attending physician. Atherapeutically effective amount of the antibody is that which provideseither subjective relief of a symptom(s) or an objectively identifiableimprovement as noted by the clinician or other qualified observer. Thesecompositions can be administered in conjunction with another agent, suchas an anti-viral agent, either simultaneously or sequentially. Theantibodies also can be used or administered as a mixture, for example inequal amounts, or individually, provided in sequence, or administeredall at once.

Single or multiple administrations of the compositions are administereddepending on the dosage and frequency as required and tolerated by thesubject. The composition should provide a sufficient quantity of atleast one of the antibodies disclosed herein to effectively treat thesubject or inhibit the development of JEV infection. The dosage can beadministered once but can be applied periodically until either atherapeutic result is achieved or until side effects warrantdiscontinuation of therapy. In one example, a dose of the antibody isinfused for thirty minutes every other day. In this example, about oneto about ten doses can be administered, such as three or six doses canbe administered every other day. In a further example, a continuousinfusion is administered for about five to about ten days. The subjectcan be treated at regular intervals, such as monthly, until a desiredtherapeutic result is achieved. Generally, the dose is sufficient totreat or ameliorate symptoms or signs of a JEV infection withoutproducing unacceptable toxicity to the patient.

In one embodiment, the antibody is a humanized monoclonal antibody thatbinds JEV, wherein the antibody has a variant Fc region, and is used forpassive immunization against a disease complicated by antibody-dependentenhancement. Antibody-dependent enhancement, a phenomenon in which viralreplication is increased rather than decreased by immune sera, has beenobserved for a large number of viruses of public health importance,including flaviviruses, coronaviruses, and retroviruses.

For passive immunization with an antibody, about 5 mg/kg to 250 mg/kg(for instance, 50-100 mg/kg) of humanized antibody is an initialcandidate dosage for administration to the subject, whether, forexample, by one or more separate administrations, or by continuousinfusion. A typical daily dosage might range from about 5 mg/kg to 250mg/kg or more, depending on the factors mentioned above. In general, itis desirable to provide the subject with a dosage of antibody which isin the range of from about 5 mg/kg-50 mg/kg, 50 mg/kg-100 mg/kg, 100mg/kg-300 mg/kg (body weight of recipient), although a lower or higherdosage can be administered. Dosages as low as about 5 mg/kg can beexpected to show some efficacy. Additionally, a dosage of about 10 mg/kgis an acceptable dose, although dosage levels up to about 250 mg/kg arealso effective, especially for therapeutic use. For repeatedadministrations over several days or longer, depending on the condition,the prophylaxis or treatment is sustained until a desired suppression ormodification of disease symptoms occurs. However, other dosage regimenscan be useful. The progress of this therapy is easily monitored byconventional techniques and assays.

The humanized antibody composition will be formulated, dosed, andadministered in a fashion consistent with good medical practice. Factorsfor consideration in this context include the particular disorder beingprevented or treated, the particular subject being treated, the clinicalcondition of the individual subject, the cause of the disorder, whetherJEV infection is present or the subject is at risk of exposure to JEV,the site of delivery of the agent, the method of administration, thescheduling of administration, and other factors known to medicalpractitioners. The prophylactically or therapeutically effective amountof the humanized antibody to be administered will be governed by suchconsiderations, and is the minimum amount necessary to prevent,ameliorate, or treat a disease or disorder. The humanized antibody canformulated with one or more anti-viral agents. The effective amount ofsuch other agents depends on the amount of humanized antibody present inthe formulation, the type of disorder or treatment, and other factorsdiscussed above.

IX. Diagnostic Methods and Kits

A method is provided herein for the detection of the expression of JEVin vitro or in vivo. In one example, expression of JEV is detected in abiological sample. The sample can be any sample, including, but notlimited to, tissue from biopsies, autopsies and pathology specimens.Biological samples also include sections of tissues, for example, frozensections taken for histological purposes. Biological samples furtherinclude body fluids, such as blood, serum, plasma, sputum, spinal fluidor urine. A biological sample is typically obtained from a mammal, suchas a rat, mouse, goat, pig, bird, horse, or primate. In one embodiment,the primate is macaque, chimpanzee, cynomogous, or human.

In several embodiments, a method is provided for detecting a JEVinfection. Blood samples from a subject suspected of having a JEVinfection contain detectable amounts of JEV protein. Thus, JEV-specificantibodies can be used to detect JEV in a blood sample from a subject todetect JEV infection in the subject, or confirm a diagnosis of JEVinfection in a subject.

The disclosure provides a method for detecting JEV in a biologicalsample, wherein the method includes contacting a biological sample witha humanized antibody that binds JEV under conditions conducive to theformation of an immune complex, and detecting the immune complex, todetect the JEV in the biological sample. In one example, the detectionof JEV in the sample indicates that the subject has a JEV infection. Inanother example, detection of JEV in the sample confirms a diagnosis ofJEV infection in a subject.

In one embodiment, the humanized antibody that specifically binds JEV isdirectly labeled with a detectable label. In another embodiment, thehumanized antibody that specifically binds JEV (the first antibody) isunlabeled and a second antibody or other molecule that can bind thehumanized antibody that specifically binds JEV is labeled. As is wellknown to one of skill in the art, a second antibody is chosen that isable to specifically bind the specific species and class of the firstantibody. For example, if the first antibody is a humanized IgG, thenthe secondary antibody can be an anti-human-IgG. Other molecules thatcan bind to antibodies include, without limitation, Protein A andProtein G, both of which are available commercially.

Suitable labels for the antibody or secondary antibody are describedabove, and include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, magnetic agents and radioactivematerials. Non-limiting examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase. Non-limiting examples of suitable prosthetic groupcomplexes include streptavidin/biotin and avidin/biotin. Non-limitingexamples of suitable fluorescent materials include umbelliferone,fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin. Anon-limiting exemplary luminescent material is luminol; a non-limitingexemplary a magnetic agent is gadolinium, and non-limiting exemplaryradioactive labels include ¹²⁵I, ¹³¹I, ³⁵S or ³H.

In an alternative embodiment, JEV can be assayed in a biological sampleby a competition immunoassay utilizing JEV standards labeled with adetectable substance and an unlabeled humanized antibody thatspecifically binds JEV. In this assay, the biological sample, thelabeled JEV standards and the humanized antibody that specifically bindJEV are combined and the amount of labeled JEV standard bound to theunlabeled antibody is determined. The amount of JEV in the biologicalsample is inversely proportional to the amount of labeled JEV standardbound to the antibody that specifically binds JEV.

The immunoassays and methods disclosed herein can be used for a numberof purposes. In one embodiment, the humanized antibody that specificallybinds JEV can be used to detect the production of JEV in cells in cellculture. In another embodiment, the antibody can be used to detect theamount of JEV in a biological sample. Increased expression of JEV isassociated with the severity of JEV infection. In one embodiment, a kitis provided for detecting JEV in a biological sample, such as a bloodsample or tissue sample. For example, to confirm a JEV infectiondiagnosis in a subject, a blood sample can be obtained to detect thepresence of JEV protein. Kits for detecting a polypeptide will typicallyinclude a humanized antibody that specifically binds JEV, such as any ofthe antibodies disclosed herein. In some embodiments, an antibodyfragment, such as an Fv fragment or a Fab is included in the kit. In afurther embodiment, the antibody is labeled (for example, with afluorescent, radioactive, or an enzymatic label).

In one embodiment, a kit includes instructional materials disclosingmeans of use of an antibody that specifically binds JEV. Theinstructional materials can be written, in an electronic form (such as acomputer diskette or compact disk) or can be visual (such as videofiles). The kits can also include additional components to facilitatethe particular application for which the kit is designed. Thus, forexample, the kit can additionally contain means of detecting a label(such as enzyme substrates for enzymatic labels, filter sets to detectfluorescent labels, appropriate secondary labels such as a secondaryantibody, or the like). The kits can additionally include buffers andother reagents routinely used for the practice of a particular method.Such kits and appropriate contents are well known to those of skill inthe art.

In one embodiment, the diagnostic kit includes an immunoassay. Althoughthe details of the immunoassays can vary with the particular formatemployed, the method of detecting JEV in a biological sample generallyincludes the steps of contacting the biological sample with an antibodywhich specifically reacts, under immunologically reactive conditions, toa JEV polypeptide. The antibody is allowed to specifically bind underimmunologically reactive conditions to form an immune complex, and thepresence of the immune complex (bound antibody) is detected directly orindirectly.

Any of the humanized antibodies that specifically bind JEV, as disclosedherein, can be used in these assays. Thus, the antibodies can be used ina conventional immunoassay, including, without limitation, an ELISA, anRIA, FACS, tissue immunohistochemistry, Western blot orimmunoprecipitation.

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the disclosure to the particular features or embodimentsdescribed.

EXAMPLES

Disclosed herein are humanized antibodies that specifically bind JEVand/or a JEV envelope polypeptide. These antibodies can be used todetect a JEV infection, or can be used to passively immunize a subjectagainst JEV.

Like other flaviviruses, JEV contains a single-stranded RNA genome thatcodes for the three virion proteins, for instance, the capsid (C),pre-membrane/membrane (prM/M) and envelope (E) proteins and sevennon-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5). TheE protein is the major protective antigen, eliciting neutralizingantibodies that play an important role in protective immune responses.In the replication cycle, the E protein mediates virus attachment toputative cell receptor(s) and viral fusion with the endosomal membranes.Three-dimensional structures of several flavivirus E's have beendetermined by x-ray crystallography (Kanai et al., (2006) J Virol80:11000-8; Modis et al., (2003) Proc Natl Acad Sci USA 100:6986-91;Modis et al., (2005) J Virol 79:1223-31; Rey et al., (1995) Nature375:291-8). The head-to-tail dimers of E are tightly organized on thevirion surface. The monomeric E is folded into three structurallydistinct domains (domain Domain III adopts an immunoglobulin-likestructure consisting of seven anti-parallel β-strands. This domain islinked by a flexible region to domain I, which folds into aneight-stranded anti-parallel β-barrel. Domain I contains approximately120 amino acids in three segments disrupted by two inserts in the formof looped sequences, which together form the dimerization domain (domainII). At the distal end of one of these domain II inserts is aflavivirus-conserved peptide shown to be involved in membrane fusion(Rey et al., (1995) Nature 375:291-8; Kuhn et al., (2002) Cell108:717-25; Rey et al., (1995) Nature 375:291-8).

Studies of mouse monoclonal antibodies from flavivirus infections haveprovided much information about E functional specificities and antigenicstructures. A majority of cross-reactive, weakly- to non-neutralizingantibodies react with epitope determinants involving the fusion peptidein domain II (Stiasny et al., (2006) J Virol 80:9557-68). Antibodiesthat recognize domain III epitopes are type-specific and efficientneutralizers of viral infection (Oliphant et al., (2005) Nat Med11:522-30; Roehrig (2003) Adv Virus Res 59:141-75). Domain III-reactiveantibodies can neutralize the virus at an early infection steppresumably by blocking viral attachment to cell receptors or byinterfering conformational changes to E, thereby preventing membranefusion (Crill & Roehrig (2001) J Virol 75:7769-73; Nybakken et al.,(2005) Nature 437:764-9). Mouse monoclonal antibodies that neutralizeflaviviruses, such as SLEV, yellow fever virus and dengue virus at hightiters in vitro, have also been shown to mediate protection of infectionin vivo (Brandriss et al., (1986) J Gen Virol 67 (Pt 2):229-34; Roehriget al., (2001) Ann NY Acad Sci 951:286-97). In the case of JEV, studieshave shown that passive transfer of mouse MAbs can protect against priorand subsequent infection in mice, goats and monkeys (Kimura-Kuroda &Yasui (1988) J Immunol 141:3606-10; Zhang et al., (1989) J Med Virol29:133-8). However, the possible immunogenicity of these antibodieslimits their clinical utility in humans. Only relatively few monoclonalantibodies that efficiently neutralize flaviviruses and map to domain Ior II have been characterized (Daffis et al., (2005) Virology337:262-72; Holzmann et al., (1997) J Gen Virol 78 (Pt 1):31-7; McMinnet al., (1995) Virology 211:10-20; Morita et al., (2001) Virology287:417-26; Ryman et al., (1997) J Gen Virol 78 (Pt 6):1353-6).Consequently, the antigenic structures of these domains and theirinvolvement in the protective immune response remain poorly understood.

There has been a lack of primate-derived antibodies for characterizationof flavivirus antigenic epitopes discovered with mouse antibodies.However, dengue virus (DENV) type-specific and cross-reactive antibodiesrecently have been recovered from infected chimpanzees by repertoirecloning. A DENV-4-specific, highly neutralizing monoclonal antibody(5H2) has been shown to react with epitope determinants in domain I anda DENV cross-reactive antibody (1A5) was shown to react with the fusionpeptide in domain II (Lai et al., (2007) J Virol. 81:12766-74; Goncalvezet al., (2004) J Virol 78:12919-28). It was also demonstrated thatpassively transferred MAb 1A5, which shares characteristics with a majorsubset of flavivirus cross-reactive antibodies, up-regulates denguevirus replication by a mechanism of antibody-dependent enhancement(Goncalvez et al., (2007) Proc Natl Acad Sci USA 104:9422-7.). Passivetransfer with the highly-neutralizing antibody MAb 5H2 has been shown toprotect mice and monkeys against DENV-4 challenge (Lai et al., (2007) JVirol. 81:12766-74).

Disclosed herein is the repertoire cloning, epitope mapping andfunctional characterization of JEV-neutralizing MAbs from immunizedchimpanzees. Several panning strategies were applied to recover Fabsthat bind to epitopes in different antigenic domains. RepresentativeMAbs that neutralized JEV efficiently and mapped to each of the threedomains in E were selected for analysis of binding activities for JEVand evaluation of their in vitro neutralizing titers against strainsbelonging to the four JEV genotypes. As proof of concept, the protectivecapacities of these humanized antibodies were analyzed in a mouse modelof Japanese encephalitis.

Example 1 Materials and Methods

This Example describes materials and methods that were used inperforming Examples 2-10. Although particular methods are described, oneof skill in the art will understand that other, similar methods also canbe used.

Viruses and Cultured Cells

Simian Vero cells and mosquito C6/36 cells were grown in MinimumEssential Medium (MEM). Schneider's Drosophila Line 2 (S2) cells werecultured in Schneider's Drosophila medium and human embryonic kidney 293T cells were cultured in Dulbecco's Modified Essential Medium (DMEM).All media were supplemented with 10% fetal bovine serum (FBS), 0.05mg/ml gentamycin, and 2.5 units/ml fungizone. Media were purchased fromInvitrogen (Carlsbad, Calif.), and cells were from the American TypeCulture Collection (Manassas, Va.). The inactivated JEV vaccine,JE-VAX®, was obtained from Sanofi Pasteur Inc. (Swiftwater, Pa.). Theattenuated JEV SA14-14-2 strain was provided by K. Eckels and R. Putnak.The JEV stock used for infection of chimpanzees, phage library panning,and plaque reduction neutralization tests (PRNT) was prepared frominfected C6/36 cells grown in VP-SFM medium (Invitrogen). The virustiter was approximately 10⁸ focus forming units (FFU)/ml as determinedon Vero cell monolayers. PRNT using the four genotype strains of wildtype JEV was performed at the Center for Vaccine Development, MahidolUniversity (Nakhonpathom, Thailand). These strains were JE 1991(genotype I), JE B1034/8 (genotype II), Beijing (genotype III) and JKT9092 (genotype IV). The JEV prototype strain Nakayama, belonging togenotype II, was used for mouse challenge experiments performed atAdimmune Corporation (Taichung, Taiwan). Experiments to detectantibody-binding specificities were performed by ELISA with DENV-1(Hawaii), DENV-2 (New Guinea B), DENV-3 (H87), DENV-4 (814669), Langatvirus (LGTV) strain TP 21, and WNV/DENV-4 chimera as describedpreviously (Goncalvez et al., (2004) J Virol 78:12910-8).

Antibodies

Humanized MAbs 1A5 and 5H2 derived from chimpanzee Fabs were prepared bytransient transfection of 293 T cells (Goncalvez et al., (2004) J Virol78:12910-8; Men et al., (2004) J Virol 78:4665-74; Kemp Biotechnology,Gaithersburg, Md.). Hyperimmune mouse ascites fluid (HMAF) raisedagainst JEV was purchased from American Type Culture Collection(Manassas, Va.). Mouse JEV complex-reactive MAb 8743 (MAb 6B4A-10) waspurchased from Chemicon (Temecula, Calif.). JEV E domain III-specificmouse MAb E3.3 was provided by S-C Wu (Lin et al., (2003) J Virol77:2600-6).

JEV E Antigen Preparations

Three different E antigen preparations from JEV SA14-14-2 were used: (i)JEV virions; (ii) domain III-specific E; and (iii) N-terminal 80% E. Toprepare JEV virions, mosquito C6/36 cells grown in MEM plus supplementswere infected with the virus at 0.1 multiple of infection (MOI) inVP-SFM medium (Invitrogen, Carlsbad, Calif.), and incubated at 32° C.The culture medium was harvested eight days after infection and keptfrozen at −80° C. The virus preparation was used for panning, ELISA andneutralization assays, as well as for selection of neutralization-escapevariants. The recombinant domain III-specific E was constructed for useas panning antigen. The protein was expressed in bacteria with ahistidine tag, essentially as described (Jaiswal et al. (2004) ProteinExpr Purif 33:80-91; Wu et al., (2003) Vaccine 21:2516-22). The DNAsequence corresponding to amino acids 296-398 (DIII) near the C-terminusof E was amplified by PCR from the viral cDNA of JEV SA 14-14-2. The DNAproduct was then purified, digested with EcoRI and HindIII, followed byinsertion into the pET21 cloning vector (Novagen, Madison, Wis.).Escherichia coli (strain BL21 (DE3)) was transformed with pET21 plasmidcontaining the insert. The histidine-tagged, domain III E protein wasaffinity-purified through a column of TALON® Metal Affinity Resin(Clontech, Mountain View, Calif.). Western blot analysis and ELISA wereperformed using JEV HMAF and MAb E3.3 to confirm the identity and properfolding of the recombinant domain III E protein.

Recombinant 80% E was generated in Drosophila S2 cells essentially asdescribed in Ledizet et al., (2005) Vaccine 23:3915-24; Men et al.,(1991) J Virol 65:1400-7; Putnak et al., (2005) Vaccine 23:4442-52. TheDNA encoding amino acids 131-692 of the PrM/N-terminal 80% E fusionprotein was amplified by PCR from JEV cDNA using the primersGGAGCCATGAAGAGATCTAATTTCCAGGGG (SEQ ID NO: 7) andGCCCAGCGTGCTCCGCGGTTTGTGCCAATGGTG (SEQ ID NO: 8). The DNA product wasdigested with BglII and SacII and inserted into the pMTBiP/V5-HisBexpression vector (Invitrogen, Carlsbad, Calif.). The recombinantplasmid and a blasticidin-resistance plasmid, pCoBlast wereco-transfected into Drosophila S2 cells according to DrosophilaExpression System Kit (Invitrogen, Carlsbad, Calif.). Stably-transformedcells were selected with blasticidin and then transferred to DrosophilaSerum-Free Medium (Invitrogen, Carlsbad, Calif.). Cultured S2 cellsexpressing JEV prM-80% E were induced with CuSO₄ at 500 μM. The secreted80% E protein is immediately followed by the V5 epitope flag andpoly-histidine tag encoded by the plasmid vector. The recombinant Eprotein was affinity-purified with TALON® Metal Affinity Resin. Westernblot analysis and ELISA were performed with HMAF, MAb E3.3 and MAb 8743to verify the identity of recombinant E. Variants of recombinant 80% Econtaining single amino acid substitutions were constructed usingQuikChange® Site-Directed Mutagenesis Kit (Stratagene, La Jolla,Calif.).

Immunization of Chimpanzees with JEV Vaccines and Construction of γ1/κAntibody Library

Two chimpanzees (96A007 and 1620) were administered subcutaneously (sc)three doses of JE-VAX® of 1 ml each at days 0, 7, and 30, according tothe regimen indicated. One year later the chimpanzees were infected witha mixture of attenuated JEV strain SA14-14-2 and WNV/DENV-4 chimera eachat 10⁶ FFU, diluted in MEM plus 0.25% human serum albumin to boost theantibody response. Eight weeks after infection, bone marrow wasaspirated from each chimpanzee and the lymphocytes were prepared bycentrifugation on a Ficoll-Paque gradient. Repertoire cloning ofchimpanzee Fab fragments was performed as previously described (Men etal., (2004) J Virol 78:4665-74). Approximately 1×10⁷ bone marrowlymphocytes from chimpanzee 96A007, which developed a higherJEV-neutralizing antibody titer than did chimpanzee 1620, were used forphage library construction. A library with a diversity of 2×10⁸˜1×10⁹was obtained at each cloning step.

Panning of Phage Library and Selection of JEV-Specific Fabs

The pComb 3H DNA library that contained the V_(L)-C_(L) and V_(H)-C_(H1)inserts was used for phage preparation as previously described (Men etal., (2004) J Virol 78:4665-74). To increase the possibility ofrecovering antibodies against different epitopes on the JEV E, threedifferent panning strategies were used. The phage library was firstpanned using JEV virions captured by chimpanzee convalescent sera coatedon the wells of an ELISA plate. Panning of the phage library by epitopemasking also was conducted as described (Ditzel et al., (1995) J Immunol154:893-906). Briefly, wells of a microtiter plate coated with JEVvirions were incubated with purified Fab A3 (isolated in panningdescribed above) at a concentration of 50 μg/ml for 1 hour at 37° C.One-fourth of volume was removed before adding 50 μl of the phagelibrary. The third strategy of antibody selection was performed usingdomain III-specific E as panning antigen. Briefly, wells of a 96-wellELISA plate were coated with 5 μg/well of purified domain III E in 0.1 Mcarbonate buffer, pH 9.0. After washing with phosphate buffered saline(PBS), antigen-coated wells were blocked with 3% bovine serum albumin(BSA). The phage library was then added as described. Following threecycles of panning in each case, the selected phage population was usedfor infection of E. coli XL-1 to produce phagemid DNA. Phagemid DNA wascleaved with SpeI and NheI to remove the phage gene III segment andcircularized for transformation of E coli XL-1. Transformed E. colicolonies were screened by ELISA to identify clones producing soluble Fabfragments reactive with JEV. Individual Fabs were prepared and screenedfor binding specificity to JEV virions or domain III E. Plasmids weresequenced to identify Fab clones with distinct V_(H) and V_(L) DNAinserts.

Production of Fabs and Humanized MAbs

The histidine-tagged Fab produced in E. coli was affinity-purified usingTALON® Metal Affinity Resin. The Fab purity was analyzed by SDS-PAGE andthe concentration determined using BCA Protein Assay Kit (Pierce,Rockford, Ill.). Construction of plasmids for expression of full-lengthhumanized IgG1 (designated as MAb thereafter) from cloned Fab DNA wascarried out as described (Men et al., (1991) J Virol 65:1400-7). MAbexpression was verified by transfection of 293T cells (purchased fromATCC) in the presence of Lipofectamine (Invitrogen, Carlsbad, Calif.)and grown in OPTIMEM® medium. One day after transfection, cells werewashed and DMEM was added. Cells were incubated for 5-7 days and theculture medium was harvested. The medium was concentrated and the MAbproduct was purified on a protein A column (Pierce, Rockford, Ill.).Scale-up MAb production was performed by Kemp Biotechnology(Gaithersburg, Md.).

Measurement of Neutralizing Titers of Fab and MAb

The neutralizing titer of Fab or MAb was determined by PRNT against therepresentative JEV strains essentially as described (Men et al., (1991)J Virol 65:1400-7; Okuno et al., (1985) Brief report. Arch Virol86:129-35). Virus foci that formed on the cell monolayer wereimmuno-stained and the antibody PRNT₅₀ titer in μg/ml was calculated.

Biotinylation of Purified Fab and Competition ELISA

Purified Fabs were biotinylated with EZ-Link NHS-LC-Biotin (Pierce,Rockford, Ill.) and used in competition ELISA. Briefly, biotin-labeledFab at a fixed concentration was mixed with dilutions of a crude orpurified preparation of competing Fab. The mixture was added to JEVvirion-coated wells and incubated at 37° C. After washing,streptavidin-alkaline phophatase (Pierce, Rockford, Ill.) was added todetect the amount of biotinylated Fab attached to the virus.

Measurement of Binding Affinity

Affinity binding analysis by ELISA or surface plasmon resonance (SPR)biosensor was performed to determine the Fab or MAb binding activity forJEV virions. ELISA was performed as described previously with minormodifications, i.e., in the absence of detergent at all steps (11, 47).JEV HMAF was used to coat the microtiter plate. Following blocking with3% BSA, JEV at a pre-determined concentration was added and incubated at37° C. for 1 hour. Dilutions of affinity-purified Fab were added andincubated at 37° C. for 1 h. The Fab bound to JEV on the microtiterplate was detected using a goat anti-human IgG-alkaline phosphataseconjugate (Sigma, St. Louis, Mo.). The steady-state equilibrium affinityconstant (K_(D)) was calculated as the Fab concentration that produced50% maximum binding.

The SPR biosensor experiments were conducted using a Biacore 3000instrument (Biacore Inc, Piscataway, N.J.) with short carboxy-methylateddextran sensor surfaces (CM3; GE Healthcare, Piscataway, N.J.) andstandard amine coupling as described (Schuck et al., (1999) Currentprotocols in protein science. New York: John Wiley & Sons2:20.2.1-20.2.21). Since the recombinant E protein showed self bindingin preliminary experiments, the E protein was immobilized on the chipsurface and the kinetics of Fab binding and dissociation were recordedfor 40 to 50 minutes and 2 hours to 10 hours, respectively, at variousFab concentrations (Schuck (1997) Annu Rev Biophys Biomol Struct26:541-566). Analysis of antibodies was conducted at a flow rate of 2μl/min for Fab B2 and 5 μl/min for Fabs A3 and E3, using PBS-P buffer(137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2.3 mM KH₂PO4, 0.005%surfactant P20, pH 7.4) at 25° C. The chip surface was regenerated with0.05% Triton X 100/2M NaCl in the case of Fab B2. No regenerationconditions were applied with Fabs A3 and E3. The kinetic traces wereglobally fitted with a model for continuous ligand distributionscombined with a two-compartment approximation of mass transport (Svitelet al., (2007) Biophys J 92:1742-58.).

Immunoprecipitation and Western Blot Analysis

Immunoprecipitation was performed with lysates of JEV-infected mosquitocells or purified recombinant E. C6/36 cells were infected with thevirus at MOI of 1 and incubated for 5 days at 32° C. Infected cells wererinsed with PBS and added lysis buffer containing 1% NP-40, 0.15 M NaCland 0.1 M Tris, pH 7.5. The cell lysate or recombinant E was incubatedwith the test antibody for 2 hours at 4° C. A 10-μl suspension ofprotein A Sepaharose beads (Calbiochem, La Jolla, Calif.) was added andthe mixture incubated overnight at 4° C. The beads containingimmunocomplexes were collected by centrifugation and washed three timeswith the lysis buffer. The immunocomplexes were added with 4× loadingbuffer (Invitrogen, Carlsbad, Calif.) and separated by SDS-PAGE. Aftertransferring onto a nitrocellulose membrane, the E protein was detectedby a mouse or humanized anti-JEV antibody followed by anti-mouse oranti-human IgG-HRP (Pierce, Rockford, Ill.), or by a mouse anti-V5epitope MAb-HRP conjugate (Invitrogen, Carlsbad, Calif.) forchemiluminescence development (Pierce, Rockford, Ill.).

Selection of JEV Antigenic Variants

Affinity-purified Fabs A3, B2 and E3 were used for selection ofneutralization-escape mutants (Goncalvez et al., (2004) J Virol78:12919-28). Briefly, approximately 1×10⁷ FFU of parental JEV SA14-14-2were mixed with 25 μg/ml of Fab in MEM and incubated at 37° C. for 1hour. The mixture was added to the Vero cell monolayer and incubated at37° C. for 1 hour. Following removal of the inoculum, the plate wasrinsed once with PBS, refed with 3 ml of MEM containing 2% FBS and 5μg/ml of the selecting Fab, and incubated at 37° C. for 5 days.Antibody-resistant variants were isolated by plaque-to-plaquepurification on Vero cells and the individual isolates were amplified ininfected C6/36 cells in the presence of the selecting Fab. Sequenceanalysis of JEV antigenic variants was conducted as described previously(Kanai et al. (2006) J Virol 80:11000-8). The JEV E structure modelingwas performed with the crystal coordinates of WNV, accession code 2169as a template (Kimura-Kuroda & Yasui (1988) J Immunol 141:3606-10), andSwissModel (Guex & Peitsch (1997) Electrophoresis 18:2714-23; Peitsch(1996) Biochem Soc Trans 24:274-9). Graphical development was performedusing the UCSF Chimera package from the Resource for Biocomputing,Visualization, and Informatics (University of California, SanFrancisco).

Mouse Model for JEV Challenge

For analysis of efficacy, groups of 4-week-old inbred ddy mice (eithersex, n=12) were infused with 0.5 ml of MAb at doses of 200, 100, 40, 20,8, 1.6 or 0.32 μg per mouse by the intraperitoneal route (i.p.) and thecontrol group received PBS diluent only. One day later, mice in allgroups were challenged by the intracerebral (i.c.) route with a diluentcontaining 40×50% lethal dose (LD₅₀) (1.5 FFU) of JEV strain Nakayama in30 μl. The animals were monitored daily for clinical signs of infection,including ruffled hair, hunched back, paralysis and death for two weeks.When signs of encephalitic paralysis developed, mice were euthanized asthe experiment end-point. In the infection-intervention experiment bypassive antibody transfer, a single dose of test MAb at 200 μg wasadministrated by the i.p. route at days 1, 3 or 5 following i.c.inoculation of 40×LD₅₀ JEV Nakayama. Mice were monitored daily forsymptoms of encephalitis for three weeks. Student's t test was used tocompare the average survival time (AST) between the mouse groups thatreceived MAb and those that received PBS.

Example 2 Chimpanzee Antibody Response to JEV Vaccines and Isolation ofFabs

This Example describes the immunization of chimpanzees with the JEVvaccine, JE-VAX, the resulting antibody response, and the isolation ofFabs using three different panning strategies.

Two chimpanzees were initially immunized with three doses of inactivatedvaccine JE-VAX®. After two months chimpanzees 96A007 and 1620 developedonly moderate PRNT₅₀ titers against JEV SA14-14-2 (1/100 and 1/71,respectively). After inoculation with a mixture of JEV SA14-14-2 andWNV/DENV-4 chimera, high JEV-neutralizing antibody titers 1/10633 and1/3114 were detected in the serum of chimpanzees 96A007 and 1620,respectively. Chimpanzee bone marrow was aspirated eight weeks afterinfection and the cells of chimpanzee 96A007 were used for a phagelibrary construction.

Selection of Fabs from a combinatorial library with a single panningantigen often yields only a dominant antibody subset that can or can notbe neutralizing. Highly neutralizing antibodies can be present as aminor subset. Therefore, three different panning strategies wereperformed in order to assemble a collection of JEV-neutralizing Fabantibodies for further functional characterizations.

Fabs Recovered from Panning with JEV Virions (Group 1 Fabs)

The phage library was first panned with JEV virions captured bychimpanzee polyclonal sera. A total of 200 E. coli clones were screenedfor Fabs reactive to the virus. Sequence alignment of 48 positive Fabclones identified four V_(H) sequences, three of which, i.e., Fabs A3,G9 and B3, were similar but not identical (FIG. 1). These Fabs appearedto represent a dominant subset of antibodies in the library. The V_(L)sequences of these four Fab clones showed three distinct patterns.Binding assay by ELISA showed that, with the exception of Fab A3, whichwas weakly reactive to WNV (detected only at 1/10 dilution), the otherthree Fabs reacted with JEV, but not with DENV-1 to −4, WNV or LGTV.These Fabs neutralized JEV efficiently at PRNT₅₀ titers ranging from2.55 to 7.91 nM (0.12 to 0.36 μg/ml; Table 2).

TABLE 2 JEV-neutralizing Fabs recovered by different panning strategiesELISA titer (1/log₁₀ Group PRNT₅₀ titer dilution) binding of^(b):(panning antigen) Fab (nM)^(a) JEV Other flaviviruses^(c) 1 (JEVSA14-14-2) A3 2.55 ± 0.42 4.7 <1 B3 4.14 ± 0.65 4.5 <1 G9 4.38 ± 1.104.4 <1 C8 7.91 ± 3.29 4.3 <1 2 (masking with B2 0.25 ± 0.09 4.2 <1 FabA3) F1 0.41 ± 0.21 4.1 <1 F3 0.45 ± 0.22 4.2 <1 A8 >1,100 4.2 <1G1 >1,040 3.0 <1 3 (recombinant E3 84.90 ± 20.2  4.2 <1 DIII E)B12 >1,070 4.0 <1 ^(a)1 nM = ~0.046 μg/ml. ^(b)Microliter plates werecoated with virions of the indicated flavivirus (10⁶ FFU/ml). The ELISAtiter was the log₁₀ reciprocal dilution of Fab that gave an opticaldensity reading twofold or greater than the background. The initial Fabconcentration was ~100 μg/ml. ^(c)DENV-1, DENV-2, DENV-3, DENV-4, WNV,and LGTV were tested. Non-neutralizing Fabs A8, G1, and B12 wereincluded for comparison.Fabs Recovered from Panning by Epitope-Masking (Group 2 Fabs)

To increase the possibility of recovering a different subset ofantibodies binding to minor epitopes on E, Fab A3 (described above) wasused for epitope-masking in a new panning of virions. From some 200clones screened, 12 Fabs bound to the virus. Sequence analysisidentified five V_(H) sequences different from members of the group 1Fabs (FIG. 1). PRNT showed that three Fabs, i.e., B2, F1 and F3, hadhigh neutralizing activities ranging from 0.25 to 0.45 nM (0.012 to0.021 μg/ml), while Fabs A8 and G1 were not neutralizing (Table 2).Members of this Fab group bound to JEV, but not to DENV-1 through 4, WNVor LGTV as analyzed by ELISA.

Fabs Recovered from Panning with Domain III E (Group 3 Fabs)

Evidence indicates that flavivirus infections elicit a major class ofcross-reactive, but weakly neutralizing antibodies that react withepitopes involving the fusion peptide in domain II E (Stiasny et al.,(2006) J Virol 80:9557-68). Studies of cloning of DENV-neutralizingantibodies from chimpanzees (Goncalvez, et al., (2004) J Virol78:12910-8) and of cloning WNV antibodies from humans (Throsby et al.,(2006) J Virol 80:6982-92) have suggested that antibodies reactive todomain III E are rare. Nevertheless, studies on WNV-neutralizingantibodies indicate that domain III E is an antigenic target in themurine model (Oliphant et al., (2005) Nat Med 11:522-30). The thirdstrategy to recover chimpanzee antibodies against JEV used domainIII-specific E as the panning antigen. Twenty-three Fabs were identifiedand sequence analysis revealed two distinct V_(H) segments, as presentin Fabs E3 and B12, with Fab E3 representing 78% of the clones. BothFabs were JEV specific. Fab E3 neutralized the virus, at a relativelylow titer (84.9 nM), compared to the neutralizing titers of Fabsselected with the previous panning strategies (Table 2). Fab B12 did notneutralize JEV (>1070 nM).

Human Homologs of Chimpanzee Antibodies

A search for sequence homology in the data base showed the most relatedhuman IgG gene homologs of the panel of chimpanzee Fabs (Table 3). Theγ1 heavy chain sequences of these Fabs demonstrated similarity to thehuman VH1, VH3, VH4 or VH7 gene families with sequence homologiesranging from 67 to 83%, excluding the CDR-3 regions. The κ light chainsequences exhibited most identity with human VK1, VK2 or VK3 genefamilies with sequence homologies of 80-95%, excluding the CDR-3regions. The four Fabs in Group I were most related to VH1 and VK1 germline genes. The γ1 heavy chain sequences of the most highly neutralizingFabs in Group II had the most identity with the human VH3 gene family.

TABLE 3 Sequence similarities between chimpanzee Fabs and their mostclosely related human immunoglobulin homologs Comparison with humanhomolog V_(H) V_(L) Chimpanzee Family % Iden- Family % Fab (gene)^(a)tity^(b) (gene)^(a) Identity^(b) Group 1 A3 VH1 (VH1-69) 70 VK1 95 G9VH1 (VH1-69) 70 VK1 80 B3 VH1 (VH1-69) 70 VK1 (VK1D-16) 91 C8 VH1(VH1-69) 67 VK1 (VK1D-27) 92 Group 2 G1 VH3 (3-49RBm) 81 VK2 (VK2-28) 87A8 VH3 (VH3-74) 82 VK1 (VK1-9) 95 B2 VH7 (VH7-4-1) 83 VK1 (VK1D-16) 86F1 VH7 (VH7-4-1) 79 VK1 (VK1D-39) 85 F3 VH7 (VH7-4-1) 82 VK3 83 Group 3B12 VH3 (VH3-74) 77 VK1 (KV1-9) 86 E3 VH4 74 VK3 (KV3D-7) 90 ^(a)TheDNAPLOT program was used to search for the most closely related homologsof human germ line IgG genes in the database. ^(b)The percent amino acididentity in the V_(H) or V_(L) segment, excluding the CDR-3 region, wasdetermined with the MEGA program.

Example 3 Fab Binding Sites on JEV by Competition ELISA

This Example describes an analysis of the relatedness of the Fab bindingsites on JEV using competition ELISA. Highly neutralizing Fabs A3, B2recognized non-overlapping epitopes on JEV.

Six Fabs that were distinct in their CDR-3H sequences were selected foranalysis of the relatedness of their binding sites on JEV by competitionELISA (FIG. 1). Fabs A3, B2, and E3 were representatives of the threeFab groups that neutralized JEV most efficiently. Additionally, Fabs A8and G1 (Group 2) and Fab B 12 (Group 3) were selected for analysis oftheir binding sites on JEV. Binding competition was not detected amongthese Fabs with each other, nor with DENV-4 specific Fab 5H2 as anegative control (Men et al., (2004) J Virol 78:4665-74; FIG. 2).Further, binding competition was not observed with Fab 1A5, a flavivirusbroadly-reactive antibody that binds to the conserved fusion loop in Eidentified earlier (Goncalvez et al., (2004) J Virol 78:12919-28). Thus,highly neutralizing Fabs A3, B2 and E3 as well as non-neutralizing FabsA8, G1 and B12 recognized non-overlapping epitopes on JEV.

Example 4 Antigenic Specificity of Fabs

This Example describes the antigenic specificity of Fabs A3, B2, and E3,as determined by Western blot.

The antigenic specificity of Fabs A3, B2, and E3 was first determined byWestern blot analysis using their derived humanized MAbs. A lysate ofJEV-infected C6/36 cells (FIG. 3A, upper panel) or a recombinant Epreparation (FIG. 3A, lower panel) was separated by SDS-PAGE and thenblotted on a nitrocellulose membrane. MAb A3 and E3 bound to E (50 kDand a minor band at 46 kD), but no such binding was detected with MAb B2under the same conditions. The possibility that MAb B2 reacts with aconformational epitope was investigated further by immunoprecipitationof the cell lysate (FIG. 3B, upper panel) or the recombinant E (FIG. 3B,lower panel) under native conditions, for instance, in the absence ofSDS and β-mercapto-ethanol. Binding of E by MAbs A3 and E3 was detectedin both panels. By comparison, the binding activity of MAb B2 for virionE as well as recombinant E was low but definitely detected, indicatingthat MAb B2 reacted with native JEV E. All three MAbs failed toprecipitate E under reducing conditions.

Example 5 Binding Activities of Fabs and Derived MAbs for JEV

This Example describes the determination of the binding activities ofthe disclosed Fabs and their respective MAbs as measured by ELISA andSPR. The order of Fab binding affinity for recombinant E was E3>A3>B2measured by SPR, whereas that for the virion E was B2>A3>E3 by ELISA.

The binding activities of Fabs and their derived MAbs for JEV virions inthe absence of detergents were determined by ELISA. The concentration ofeach antibody required to attain 50% maximum binding was calculated bynonlinear regression (FIG. 4A). The concentration provides an estimateof the binding affinity K_(D). Accordingly, the K_(D) was 0.45±0.06 nMfor Fab A3, 0.28±0.11 nM for Fab B2 and 0.98±0.07 nM for Fab E3 (Table4). Conversion from the monovalent Fab to the bivalent MAb formincreased the antibody avidity 3- to 4-fold. A consistent correlationwas observed for each antibody when the K_(D) value was compared withthe neutralization titer (r=0.97).

Next, it was determined whether there is a similar correlation betweenFab binding affinity for recombinant E and the neutralization potency.SPR measurements allow a precise, real-time determination of Fab-Eassociation and dissociation rates. Representative tracings for Fabs A3and B2 are shown in FIG. 4B. Using this analysis, the affinity constantK_(d) measured for Fab A3 was 0.72 nM and that for Fab E3 was 0.35 nM,comparable to the K_(D) values measured by ELISA (Table 4).Surprisingly, the Kd of Fab B2 for binding to recombinant E was ≧150fold weaker than that measured for Fab A3 or E3. The Kd of Fab B2 wasalso significantly weaker than the K_(D) measured with virions by ELISA(110 nM vs 0.15 nM, respectively). The off-rate of Fab B2 was ≧120-foldfaster than that of Fab A3 or E3. The order of Fab binding affinity forrecombinant E was E3>A3>B2 measured by SPR, whereas that for the virionE was B2>A3>E3 by ELISA.

TABLE 4 Binding and neutralizing activities of Fabs and MAbs SPR^(a)ELISA, KD (nM)^(b) Antibody kon (M⁻1 s⁻1) koff (s⁻1) Kd (nM) Fab MAbPRNT₅₀(nM)^(c) A3 4.0 × 10⁴ 2.8 × 10⁻⁵ 0.72 0.45 ± 0.06 0.15 ± 0.07 2.55± 0.42 B2 4.2 × 10⁴ 4.6 × 10⁻³ 110 0.28 ± 0.11 0.08 ± 0.03 0.25 ± 0.09E3 1.0 × 10⁵ 3.6 × 10⁻⁵ 0.35 0.98 ± 0.07 0.31 ± 0.10 84.9 ± 20.2^(a)Recombmant E was immobilized on the solid phase, and real-timemeasurements of Fab binding were made by SPR. ^(b)Concentration of Fabor MAb that gave half-maximal binding to JEV virions determined byELISA. ^(c)Concentration of Fab that neutralized 50% of JEV plaques onVero cells.

Example 6 Localization of Epitope Determinants on E

This Example describes mapping of the epitope determinants ofJEV-neutralizing antibodies by determining the C-prM-E sequences ofvariants v1, v2 and v3 and the parental virus.

Fabs A3, B2, and E3 were each used to isolate neutralization-escapemutants of JEV SA-14-14-2. Antigenic variant JEV-v1 was isolated fromFab A3, JEV-v2 from Fab B2 and JEV-v3 from Fab E3. When the selectingFab was used in the neutralization assay, variants JEV-v1 and JEV-v2showed approximately 340- and 132-fold more resistance than the parentalvirus, respectively (FIGS. 5A and 5B). JEV-v3 was completely resistantto neutralization by 1080 nM Fab E3 (FIG. 5C).

To map the epitope determinants of JEV-neutralizing antibodies, theC-prM-E sequences of variants v1, v2 and v3 and the parental virus weredetermined. FIG. 6A shows the sequence alignment in the regionssurrounding the amino acid substitutions in the variants. Variant v1contained two substitutions in E, Lys₁₃₆-Asn (β-strand E₀) andLys₁₇₉-Glu (β-strand Go), both located in domain I. These two aminoacids are conserved, but the surrounding amino acids vary among membersof the JE group, which possibly accounts for the lack of reactivity ofFab A3 with WNV by ELISA (Table 2). These amino acids were 20.7 Å apartand exposed on the surface of E, according to the 3-D JEV E proteinmodel based on the WNV E crystal coordinates (FIG. 6B). Variant v2 alsocontained two substitutions, Ile₁₂₆-Thr (β-strand e) and Tyr₂₁₉-His(α-A), at a distance of 16.2 Å in domain II. Binding of Fab B2 to WNVwas not observed, despite the conservation of Ile₁₂₆ and surroundingamino acids (FIG. 6A). Variant v3 also contained two substitutions,Gly₃₀₂Asp in domain III and Ile₁₂₆Thr in domain II. The two positionswere approximately 64.6 Å apart and Fab E3 reacted with domain IIIsequences, indicating that Ile₁₂₆Thr was not responsible for resistanceto neutralization by this antibody. Based on the comparison of theescape variant and the wild type virus PRNT₅₀ titers, it is possiblethat there might be other major epitope determinants, especially forFabs A3 and B2.

Example 7 JEV Recombinant Es Containing Single Amino Acid Substitutions

This Example describes analysis of the effect of mutations on antibodybinding. Lys₁₃₆ in domain I, Ile₁₂₆ in domain II, and Gly₃₀₂ in domainIII are the major epitope determinants of MAbs A3, B2 and E3,respectively.

Since there were two mutations in E of each JEV variant, the effect ofeach mutation on antibody binding was analyzed. JEV E proteinscontaining a single substitution of Ile₁₂₆-Thr, Lys₁₃₆-Asn, Lys₁₇₉-Asp,His₂₁₉-Tyr or Gly₃₀₂-Asp were generated. Immunoblots showed Lys₁₃₆-Asnsubstitution had no effect, whereas the Lys₁₇₉-Asp mutation lostreactivity for MAb A3 (FIG. 7A). Similarly, Gly₃₀₂-Asp substitution lostreactivity for MAb E3, whereas Ile₁₂₆-Thr had no effect, as predicted.The antibody-binding patterns of these E constructs were also confirmedby immunoprecipitation (FIG. 7B). The latter assay further showed thatIle₁₂₆-Thr substitution lost the reactivity for MAb B2, whereasHis₂₁₉-Tyr did not affect binding. As a positive control, mouse MAb6B4A-10 reacted with all of these mutant E constructs in both assays.These results support the conclusion that Lys₁₃₆ in domain I, Ile₁₂₆ indomain II, and Gly₃₀₂ in domain III are the major epitope determinantsof MAbs A3, B2 and E3, respectively.

Example 8 Neutralization of the Attenuated and Wild Type JEV Strains byFabs and Humanized MAbs

This Example describes neutralization of the attenuated JEV strain andthe wild type strains representing each genotype by threehighly-neutralizing Fabs and humanized MAbs derived from these Fabs.

Earlier the neutralizing titers of Fabs were determined using attenuatedJEV strain SA14-14-2 (Genotype III). Three highly-neutralizing Fabs werefurther evaluated for neutralization of wild type JEV strainsrepresenting each of the four genotypes. Each of these Fabs neutralizedwild type members of Genotype I-IV as efficiently as the attenuatedstrain, with the exception that Fab B2, which neutralized strain JKT9092 (Genotype IV) at a PRNT₅₀ titer reduced by greater than 10³ fold(Table 5). Fab B2 was the most efficient neutralizer of other strainsand Fab E3 was the least efficient. Humanized MAbs derived from theseFabs were also used for neutralization of the attenuated strain and thewild type strains representing each genotype. MAbs A3 and B2 showed aPRNT₅₀ titer 3-100 fold higher than that of the Fab counterpart. MAb E3had a PRNT₅₀ titer 40 to >1000 fold higher than that measured for Fab E3against all genotype strains. JEV JKT 9092, like other strains, wasefficiently neutralized by MAb A3 and E3, although it was onlymoderately neutralized by the highly neutralizing MAb B2.

TABLE 5 Neutralization of JEV strains representing genotypes I to IV byFabs and humanized MAbs PRNT₅₀ titer (nM)^(a) of antibody: Genotype A3B2 E3 (strain) Fab MAb Fab MAb Fab MAb I (JE1991) 1.30 0.04 0.06 0.0253.05 0.21 II (JE B1034/8) 3.47 0.04 1.85 0.02 161.74 0.14 III (Beijing)2.82 0.20 0.06 0.02 27.60 0.71 III (SA14-14-2) 2.55 0.20 0.25 0.03 84.900.93 IV (9092) 1.74 0.07 >2,170 2.00 22.00 0.36 ^(a)1 nM = ~0.15 μg/ml.

Example 9 Protective Capacity of Humanized MAbs Against JEV Infection inMice

This Example describes the evaluation of the protective and therapeuticcapacity of the humanized MAbs against JEV infection in mice.

The mouse JEV encephalitis model used for validation of the inactivatedJEV vaccine in commercial production was employed to evaluate theprotective capacity of humanized MAbs. Four-week-old, inbred ddy micewere each inoculated subcutaneously with a single dose of MAb rangingfrom 0.32 to 200 μg. Mice were challenged with 40×LD₅₀ of JEV strainNakayama intracerebrally 24 hours later. At a dose of 200 μg/per mouse,Mabs A3 and B2 protected 100% and Mab E3 protected 75% of mice in thegroups, compared to no survival in the unprotected group following viruschallenge (FIG. 8). Titration of MAbs against virus infection showed adose-dependent response in terms of the survival rate and averagesurvival time. The 50% protective dose per mouse calculated according tothe ordinal-regression model probit was 0.84 μg for MAb B2, 5.8 μg forMAb A3 and 24.7 μg for MAb E3. The protective capacities of these MAbsranked in the same order as their neutralizing activities in vitro.These experiments confirm the feasibility of neutralizing MAb passivetransfer for prevention of JEV encephalitis.

The use of these MAbs for therapy of JEV encephalitis was alsoinvestigated. MAb B2 administered at a single dose of 200 μg one dayafter JEV infection resulted in a 50% survival rate (Table 6). Althoughfewer survivals were found after similar transfer with the lessprotective MAb A3 or E3 one day after JEV infection, the averagesurvival time increased significantly with MAb B2 (8.0±1.4 days) or MAbA3 (7.4±0.7 days), compared with 5.9±0.8 days for unprotected animals.Thus, passive transfer with either of these MAbs improved the outcome ofJEV infection when administered one day prior to infection (Table 6).However, the average survival time was in the range of 6.2 to 6.3 daysfor MAb A3 and 5.2 to 6.3 days for MAb B2, not significantly differentfrom 5.8 to 5.9 days for the PBS control group when administrated 3 or 5days prior to infection.

TABLE 6 Protection by passive transfer of MAb to mice against priorinfection with 40x LD₅₀ of JEV strain Nakayama 1 day earlier No.survived/no. in group MAb (AST^(a) ± SD [days]) P value A3 3/12 (7.4 ±0.7) 0.0001^(b) B2 6/12 (8.0 ± 1.4) 0.015^(b) E3 3/12 (6.3 ± 1.0) 0.22PBS 0/12 (5.9 ± 0.8) ^(a)AST, average survival time. ^(b)The averagesurvival time of the indicated MAb-treated group was significantlydifferent from that of the control group by t test. PBS diluent was usedin the group in lieu of MAb. Survival rates were also calculated.

Example 10 Humanized Monoclonal Antibodies Derived from Chimpanzee FabsEfficient for Neutralization of Japanese Encephalitis Virus (JEV) InVitro and Protection Against JEV Infection in Mice

This example describes the epitopes of the envelope protein bound bymonoclonal antibodies, including neutralizing antibodies.

Different panning strategies have served the purpose of recovering alarge panel of JEV antibodies reactive with epitopes that mapped to allthree domains in E. Fab A3 and three other Fabs that were selected forstrong binding to JEV SA14-14-2 virions were highly neutralizing (Table2). These Fabs appear to represent a major subset of neutralizingantibodies reactive to an immunodominant epitope(s) on JEV. A majordeterminant of the epitope reactive to Fab A3 mapped to Lys₁₇₉ within aβ-strand in domain I of E. Lys₁₇₉ is conserved among JEV strains ofdifferent genotypes, indicating the importance of this neutralizingantibody for protection. Lys₁₇₉ of JEV E aligned with Lys₁₇₄ in DENV-4E. Only a few mouse MAbs that neutralize flaviviruses have been shown toreact with epitope determinants that mapped to domain I in E (Holzmannet al., (1997) J Gen Virol 78 (Pt 1):31-7; Ryman et al., (1997) J GenVirol 78 (Pt 6):1353-6; Ryman et al., (1997) Virology 230:376-80). Forexample, the epitope determinants of TBEV-neutralizing MAbs i2 and IC3from mice have been mapped to positions 171 and 181 (corresponding toJEV E position 170 and 180; Holzmann et al., (1997) J Gen Virol 78 (Pt1):31-7). These results demonstrate that a cluster of epitopes involvingthe antigenic determinant Lys₁₇₉ in JEV E (or the corresponding Lys₁₇₄in DENV-4 E) apparently shared between rodents and primates (chimpanzeesand possibly humans), play an important role in inducing flavivirustype-specific antibodies.

The epitope-masking, followed by panning, has allowed the recovery ofFab B2 and two other related antibodies (Fabs F1 and F3) with highneutralizing titers against JEV in vitro. Sequence analysis of Fab B2neutralization-escape variant v2 identified Ile₁₂₆ within the small loopbetween d and e β-strands in domain II. Evidence for epitope determinantIle₁₂₆ is also supported by the demonstration that substitution of I₁₂₆Tin recombinant 80% E truncated at the C-terminus resulted in loss ofbinding for MAb B2. Epitopes that are closely related to this d-e loopepitope in domain II have been described for mouse antibodies againstJEV or other flaviviruses by analysis of antigenic variants (Hasegawa etal., (1992) Virology 191:158-65; Holzmann et al., (1997) J Gen Virol 78(Pt 1):31-7; McMinn et al., (1995) Virology 211:10-20; Morita et al.,(2001) Virology 287:417-26). For example, antigenic variants partiallyresistant to mouse JEV-neutralizing MAb 503 were found to containmutations at Ile₁₂₆ (domain II), Lys₁₃₆ (domain I) or Ser₂₇₅ (domain II)in E clustered at the junction of domains I and II. Presumably, some ofthese amino acids are contact residues for this MAb. Thus, the epitopereactive to MAb 503 appears to consist of discontinuous sequencesinvolving an important determinant at Ile₁₂₆ (Morita et al., (2001)Virology 287:417-26). These observations suggest that these chimpanzeeand mouse neutralizing MAbs react with similar or overlapping epitopes,probably involving a common determinant at or near position 126.

It is clear that JEV virions can select strongly-neutralizing antibodiesreactive to domain I- and II-specific epitopes. Two domain III-reactiveFabs were also recovered with the use of domain III-specific recombinantE. One of these (Fab E3) had a moderate neutralizing activity in vitroand its epitope determinant mapped to JEV-conserved Gly₃₀₂ within theN-terminal segment of domain III (amino acid residues 302-309). Further,Fab E3 competed with the binding of mouse MAb E3.3, which recognizes aconformation-dependent epitope in E domain III (Wu et al., (2003) J BiolChem 278:46007-13). It has been reported that the most potent flavivirusneutralizing antibodies recognize epitopes on the upper lateral surfaceof domain-III, composed of residues of the amino-terminal region and thethree loops FG, BC and DE (Nybakken et al., (2005) Nature 437:764-9;Sukupolyi-Petty et al., (2007) J. Virol 81:12816-26; Wu et al., (2003) JBiol Chem 278:46007-13). Highly neutralizing antibodies that recognizesequences in domain III were not recovered from the chimpanzee antibodylibrary. This apparent lack of immunodominance of domain III antibodieswas not surprising in view of experience with neutralizing antibodiesfrom DENV-infected chimpanzees and the recent characterization of humanantibodies against WNV (39, Throsby et al., (2006) J Virol 80:6982-92).However, it can not be ruled out that JEV virions or the recombinantdomain III E protein coated on the plate had not assumed the nativeconformation for binding to highly neutralizing antibodies.

Recent studies with DENV, WNV and TBEV suggest that a major subset ofbroadly cross-reactive antibodies are directed against immuno-dominantepitopes that include the fusion peptide in the E protein (Goncalvez etal., (2004) J Virol 78:12919-28; Stiasny et al., (2006) J Virol80:9557-68; Throsby et al., (2006) J Virol 80:6982-92). Without beingbound by theory, the most plausible explanation for the lack of suchantibodies described herein is that SA14-14-2 virus used for panningbinds weakly to the cross-reactive antibodies, thereby preventing theirisolation. The Phe₁₀₇Leu substitution in the E fusion loop alone wasresponsible for reduced binding affinity of SA 14-14-2 virions to thebroadly cross-reactive chimpanzee MAb 1A5 (Goncalvez et al., (2004) JVirol 78:12919-28).

ELISA provided useful insights into Fab binding activities for JEVvirions. Highly-neutralizing Fabs B2 and A3 reached half maximum bindingat approximately 0.5 nM and ˜10 nM respectively, whereas the comparablevalue for moderately-neutralizing Fab E3 was ˜100 nM (FIG. 4A). Theconcentration for half-maximum binding, together with the PRNT₅₀ titersallowed measurement of antibody neutralization potency, based on thecalculation of the threshold occupancy of accessible antibody sites onthe virion (Pierson et al., (2007) Cell Host & Microbe 1:135-145).According to the multiple-hit theory and stoichiometric analysis ofepitope occupancy for neutralization (Klasse et al., (2002) J Gen Virol83:2091-108), the most potent antibodies neutralize the virus atconcentrations with low occupancy of the epitopes available for bindingon the virion. The occupancy for the most potent JEV-neutralizing MAb B2was approximately 28% of available sites, whereas the occupancy for MAbsA3 and E3 were calculated at 45% and 66% of the accessible sites,respectively. The three JEV-neutralizing antibodies bind to specificepitopes in three separate E domains and most probably neutralize thevirus by different mechanisms. Other contributing factors for assessmentand interpretation of the antibody binding stoichiometry likely alsoinclude the epitope presentation of antigen preparations. To thateffect, the binding affinity of Fab B2 for the recombinant E proteinmeasured by SPR was very different from that determined for the virionby ELISA, for instance, Kd of 110 nM vs K_(D) of 0.28 nM (Table 4). Onepossible explanation is the conformational dependency of the B2 epitope,as shown by the loss of MAb B2 binding to the recombinant E or thevirion in a Western blot assay. Accordingly, the number of accessiblesites for B2 binding differed between the recombinant E and the virionon a molar basis. The high neutralization potency of MAb B2 could bepartially determined by a higher affinity for a limited subset of Eprotein conformations that most closely mimic E on the viral surface.

The presently recognized four JEV genotypes show a 7% or greaternucleotide sequence divergence based on limited sequences (Chen et al.,(1992) Am J Trop Med Hyg 47:61-9; Solomon et al., (2003) J Virol77:3091-8). Strains of genotype IV are the least similar and probablyrepresent the ancestral lineage with up to 20% nucleotide and 6.5% aminoacid divergence compared to other genotype strains. Genotypes I-III aremost wide-spread and responsible for epidemic disease. As demonstratedherein, each of the three Fabs and derived humanized MAbs exhibits ahigh neutralizing activity against a broad spectrum of JEV genotypestrains. One single exception is that the neutralizing activity of MAbB2 against JEV strain 9092 (genotype IV) was reduced by approximately100 fold, compared to that against other genotype strains. A sequencesearch of strain 9092 (accession #U70409) in the data base revealed thatthe substitution of Ile₁₂₆Thr identified earlier in the B2 escape mutantwas not present. This observation suggests the possibility that othermutations in E of the JEV strain affecting MAb B2 binding andneutralization are present in this one strain. A sequence analysis ofother genotype IV strains revealed the presence of the Ile₁₂₆Thrsubstitution in strains JKT 6468 (accession #AY184212) and JKT 7003(accession #70408) in E, indicating that both JEV strains can exhibitresistance to neutralization by MAb B2. Strains of genotype IV were allisolated in 1980-1981 from mosquitoes and are believed to have remainedin the Indonesia-Malaysia region; this substitution could be limited tothese strains.

Unlike JEV genotype IV strains, strains of genotypes I-III have spreadwidely in Asia in recent years. Immunization using the inactivated orlive SA14-14-2 JEV vaccine, both prepared from genotype III strains, haseffectively controlled JE epidemics in most countries. However, JEVoutbreaks remain a public health problem for residents in the regionswhere JEV vaccination is inadequate and a concern for travelers to theseregions as well. Antibody-mediated prevention of JEV infection is analternative to vaccines. Demonstration of passive protection withhumanized chimpanzee MAbs against JEV infection in vivo is providedherein. The 50% protective dose (ED₅₀) was measured for MAbs B2 (0.32μg), A3 (5.8 μg) and E3 (24.7 μg) for 21-g mice. It is evident thatJEV-protective efficacies in vivo correlate well with neutralizingactivities in vitro. Administration of 200 μg/mouse of MAb B2 one dayafter lethal intracerebral JEV infection protected 50% of mice, whereasall mice in the control group died. A significant improvement of JEVinfection survival time after administration of MAbs B2 and A3 was alsoevident. Thus, these MAbs are therapeutically effective.

In contrast, the average survival time was not prolonged when mice wereinoculated with any of the antibodies three or five days after JEVchallenge. Virus titers can reach ˜1×10⁷ to 1×10⁸FFU/g in the brain of3-week-old mice three to five days after intracerebral inoculation ofJEV (Kuhn et al., (2002) Cell 108:717-25). Other mouse JE encephalitismodels employing less severe intraperitoneal inoculation have also beendescribed (Jan et al., (1993) Am J Trop Med Hyg 48:412-23; Kimura-Kuroda& Yasui (1988) J Immunol 141:3606-10). Studies have shown thatinoculation of 200 μg of mouse MAb 503 on day 5 after intraperitonealchallenge protected 82% of the animals (Kimura-Kuroda & Yasui (1988) JImmunol 141:3606-10). Passive protection on three or five days afterinfection by our humanized antibodies would probably have been possibleif the virus were introduced into the animals intraperitoneally.However, the test used herein (by challenging mice intracerebrally) wasa more stringent test of protection. Infection intervention could befurther improved by the combined use of two or more Mabs, such as B2 andA3, which react to separate domains and possibly neutralize the virus bydifferent mechanisms.

Example 11 Neutralization Synergy of Humanized JEV MAbs In Vitro

This Example evaluates neutralization synergy of humanized JEV MAbs forattenuated JEV strain SA-14-14-2.

Earlier the neutralizing titers of individual MAbs were determined usingattenuated JEV strain SA-14-14-2 (Example 8). The level ofneutralization enhancement observed in vitro when combining neutralizingJEV MAbs A3, B2, and/or E3, was determined using two differentapproaches.

First, a classical approach was used, in which the MAbs were mixed at afixed (constant) ratio determined on the basis of their relativeneutralization potency (50% plaque reduction neutralization test,PRNT₅₀). Dose-response curves were determined for the antibody mixture(combination) and for each of the individual antibodies in the mixture.The presence or absence of neutralization synergy was assessed bycomparing the PRNT₅₀ values for each of the individual antibodies to theantibody mixture. No synergy was evident by comparison of the PRNT₅₀ ofeach of the individual antibodies (A3, B2, or E3) as compared to thevalues of the double (A3 and B2; B2 and E3; or A3 and E3) or triple (A3and B2 and E3) JEV MAb combination (Table 7).

TABLE 7 Neutralization of JEV SA-14-14-2 by MAbs alone or incombination^(a). Antibody PRNT₅₀ (μg/ml)^(b) A3 0.04 B2 0.005 E3 0.09A3:B2 (1:1) 0.008 A3:E3 (1:1) 0.03 B2:E3 (1:1) 0.006 A3:B2:E3 (1:1:1)0.006 ^(a)Neutralization synergy of antibody combinations for JEVSA-14-14-2 were determined using a dose-response curves for each of theantibodies alone and for antibody combinations mixed in an equimolarratio. ^(b)PRNT₅₀ were calculated by estimating the 50% neutralizationtiter from the neutralization curves.

A second approach with a variable antibody ratio was also used todetermine neutralization synergy of the JEV MAbs in vitro usingattenuated JEV strain SA14-14-2. In this approach, one antibody in thecombination was titrated and a fixed amount of a second neutralizingantibody at a weakly neutralizing concentration was added. The presenceof neutralization enhancement was assessed by comparing the PRNT₅₀values of the combination antibody mixture to those obtained with asingle antibody titration. An enhancement of neutralization wasdetermined as a significant increase of the titration curve of theantibody mixture compared to the titration curve of the first antibodyalone. The results shown in Table 8 for neutralization of JEV SA-14-14-2with MAbs A3, B2, or E3, and their combinations confirm the resultsshown in Table 7. The mixing of MAbs A3, B2, or E3 in all possible twoantibody combinations did not alter the neutralization titer observed.Thus, a combination of humanized JEV MAbs did not improve upon theneutralization titers observed for the corresponding individualhumanized JEV MAb titers (Tables 7 and 8).

TABLE 8 Neutralization of JEV SA-14-14-2 by MAbs alone or incombination^(a). PRNT₅₀ (μg/ml) of MAb 1 or in combination with MAb2^(c) MAb 2 (μg/ml)^(b) A3 B2 E3 None 0.04 0.005 0.09 A3 (0.03) — 0.0080.11 B2 (0.004) 0.05 — 0.06 E3 (0.1) 0.03 0.005 — ^(a)Neutralizationsynergy of antibody combinations for JEV SA14-14-2 were determined usingthe neutralization dose-response curve of MAb 1 in the presence orabsence of a fixed, weakly neutralizing concentration of MAb 2. ^(b)Thefixed concentration of MAb 2 is indicated in parenthesis and representsa weak neutralizing concentration. ^(c)PRNT₅₀ of MAb 1 is shown alone orin combination with a weakly neutralizing concentration of MAb 2.

Example 12 Protective and Therapeutic Capacity of Humanized MAbCombinations Against JEV Infection in Mice

This example describes the evaluation of the protective and therapeuticcapacity of the humanized MAb combination (A3 and B2) against JEVinfection in mice.

The mouse JEV encephalitis model used for validation of the inactivatedJEV vaccine in commercial production was employed to evaluate thetherapeutic capacity of the humanized MAb combination. Groups of4-week-old inbred ddy mice (either sex, n=12) were each challenged viathe intracerebral (i.c.) route with a diluent containing 40×50% lethaldose (LD₅₀) (1.5 FFU) of JEV strain Nakayama in 30 μl. One or two dayslater, mice were infused with 0.5 ml (20 μg, 100 μg or 200 μg per mouse)of MAbs A3 or B2 alone, or in combination by intraperitoneal (i.p.)route; the control group received PBS diluent only. The mice weremonitored daily for clinical signs of infection, including ruffled hair,hunched back, paralysis, and death. When signs of encephalitic paralysisdeveloped, mice were euthanized as the experiment end point. A Student'st test was used to compare the average survival times (AST) between themouse groups that received a single MAb with those that received acombination of MAbs. At a dose of 200 μg/per mouse, MAb B2 protected 33%of mice in the group, compared to no survivals in the unprotected groupfollowing MAb administration (Table 9). A dose of 20 μg/per mouse MAb A3or 100 μg/per mouse MAb A3 failed to protect any mice in these groupsfollowing MAb administration.

The use of these MAb combinations for therapy of JEV encephalitis wasalso investigated. MAb B2 administered at a single dose of 200 μg 1 dayafter JEV infection resulted in a 33% survival rate (Table 9). A slightincrease in the survival rate (41.7%) was found after transfer of acombination of MAbs B2 and A3 at a ratio of 2:1, 1 day after JEVinfection. However, the average survival time (AST) of this group wasnot significantly different from the group that received PBS (7.1±0.4days vs. 7.0±0 days; P>0.5, t test) or MAb B2 alone (7.1±0.4 days vs.10.8±5.5 days;: P>0.05, t test). Thus, passive transfer of a combinationof MAbs B2 and A3 at a ratio of 2:1 or 10:1 one day after viruschallenge did not improve the outcome of JEV infection when comparedwith the group that received MAb B2 alone (Table 9). Nevertheless, foran effective immunotherapy and/or immunoprophylaxis in humans, acombination of JEV-neutralizing, non-competing MAbs might be requiredfor control of potential neutralization escape mutants and coverage ofdifferent strains of JEV.

TABLE 9 Protection by passive transfer of MAb to mice against priorinfection with 40x LD₅₀ of JEV strain Nakayama 1 day after challenge.No. Survived/no. in group MAb (μg/mouse) (AST^(a) ± SD [days]) B2(200)4/12 (10.8 ± 5.5) B2(200):A3(100) 5/12 (7.1 ± 0.4) A3(100) 0/12 (7.3 ±0.5) B2(200):A3(20) 3/12 (7.8 ± 0.7) A3(20) 0/12 (7.0 ± 0) PBS 0/12 (7.0± 0) ^(a)AST, average survival time. PBS diluent was used in the controlgroup.

This disclosure provides humanized monoclonal antibodies specific forJEV. The disclosure further provides methods of treating, preventing, orameliorating JEV infection. It will be apparent that the precise detailsof the methods described can be varied or modified without departingfrom the spirit of the described disclosure. We claim all suchmodifications and variations that fall within the scope and spirit ofthe claims below.

The invention claimed is:
 1. A method of passively immunizing a subjectagainst a Japanese encephalitis virus, comprising selecting a subjectexposed to Japanese encephalitis virus or at risk of exposure toJapanese encephalitis virus; and administering to the subject atherapeutically effective amount of a humanized antibody or antigenbinding fragment thereof, wherein the humanized antibody or antigenbinding fragment comprises one of: (a) a heavy chain comprising aminoacids 31-35 of SEQ ID NO: 1, amino acids 50-66 of SEQ ID NO: 1, andamino acids 96-107 of SEQ ID NO: 1 and a light chain comprising aminoacids 24-34 of SEQ ID NO: 4, amino acids 50-56 of SEQ ID NO: 4, andamino acids 89-96 of SEQ ID NO: 4; (b) a heavy chain comprising aminoacids 32-36 of SEQ ID NO: 2, amino acids 50-67 of SEQ ID NO: 2, andamino acids 100-113 of SEQ ID NO: 2 and a light chain comprising aminoacids 24-34 of SEQ ID NO: 5, amino acids 50-56 of SEQ ID NO: 5, andamino acids 89-96 of SEQ ID NO: 5; or (c) a heavy chain comprising aminoacids 30-37 of SEQ ID NO: 3, amino acids 52-67 of SEQ ID NO: 3, andamino acids 100-112 of SEQ ID NO: 3and a light chain comprising aminoacids 22-33 of SEQ ID NO: 6, amino acids 49-55 of SEQ ID NO: 6, andamino acids 88-95 of SEQ ID NO: 6; thereby passively immunizing thesubject against the Japanese encephalitis virus.
 2. The method of claim1, wherein the heavy chain of the humanized antibody or antigen bindingfragment comprises amino acids 31-35 of SEQ ID NO: 1, amino acids 50-66of SEQ ID NO: 1, and amino acids 96-107 of SEQ ID NO: 1 and the lightchain of the humanized antibody or antigen binding fragment comprisesamino acids 24-34 of SEQ ID NO: 4, amino acids 50-56 of SEQ ID NO: 4,and amino acids 89-96 of SEQ ID NO:
 4. 3. The method of claim 1, whereinthe humanized antibody or antigen binding fragment is administeredintravenously or intramuscularly.
 4. The method of claim 1, wherein theheavy chain of the humanized antibody or antigen binding fragmentcomprises amino acids 32-36, 50-67 and 100-113 of SEQ ID NO: 2, and thelight chain of the humanized antibody or antigen binding fragmentcomprises amino acids 24-34, 50-56 and 89-96 of SEQ ID NO:
 5. 5. Themethod of claim 1, wherein the heavy chain of the humanized antibody orantigen binding fragment comprises amino acids 30-37, 52-67 and 100-112of SEQ ID NO: 3, and the light chain of the humanized antibody orantigen binding fragment comprises amino acids 22-33, 49-55 and 88-95 ofSEQ ID NO: 6, or an antigen binding fragment of the isolated humanizedmonoclonal antibody.
 6. The method of claim 1, wherein the heavy chainof the humanized antibody or antigen binding fragment comprises one ofSEQ ID NO; 1, SEQ ID NO: 2, or SEQ ID NO:
 3. 7. The method of claim 1,wherein the light chain of the humanized antibody or antigen bindingfragment comprises one of SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.8. The method of claim 1, wherein the heavy chain of the humanizedantibody or antigen binding fragment comprises SEQ ID NO: 1 and thelight chain of the humanized antibody or antigen binding fragmentcomprises SEQ ID NO:
 4. 9. The method of claim 1, wherein the heavychain of the humanized antibody or antigen binding fragment comprisesSEQ ID NO: 2and the light chain of the humanized antibody or antigenbinding fragment comprises SEQ ID NO:
 5. 10. The method of claim 1,wherein the heavy chain of the humanized antibody or antigen bindingfragment comprises SEQ ID NO: 3 and the light chain of the humanizedantibody or antigen binding fragment comprises SEQ ID NO:
 6. 11. Themethod of claim 1, wherein the humanized antibody or antigen bindingfragment specifically binds to Lys₁₇₉ within a β-strand in domain I ofthe envelope protein.
 12. The method of claim 1, wherein the humanizedantibody or antigen binding fragment specifically binds Ile₁₂₆ withinthe small loop between d and e β-strands in domain II of the envelopeprotein.
 13. The method of claim 1, wherein the humanized antibody orantigen binding fragment specifically binds Gly₃₀₂ within amino acids302-309 of domain III of the envelope protein.
 14. The method of claim1, wherein the antigen-binding fragment is a Fab fragment, a Fab′fragment, a F(ab)′₂ fragment, a single chain Fv protein (“scFv”), or adisulfide stabilized Fv protein (“dsFv”).
 15. The method of claim 1,wherein the antibody is a Fab fragment.
 16. The method of claim 1,wherein the humanized antibody is an IgG.
 17. The method of claim 1,wherein multiple doses of the humanized antibody or antigen bindingfragment are administered to the subject.
 18. The method of claim 1,wherein the humanized antibody or antigen binding fragment isadministered parenterally.
 19. The method of claim 1, further comprisingadministering to the subject a therapeutically effective amount of ananti-viral agent.